Ceftolozane antibiotic compositions

ABSTRACT

This disclosure provides pharmaceutical compositions comprising ceftolozane, pharmaceutical compositions comprising ceftolozane and tazobactam, methods of preparing those compositions, and related methods and uses of these compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patent application Ser. No. 14/251,381, filed Apr. 11, 2014, which is a continuation application under 35 U.S.C. 111 of International Application No. of PCT/US2014/028642, filed Mar. 14, 2014, which claims priority to U.S. Provisional Application No. 61/792,092, filed Mar. 15, 2013; U.S. Provisional Application No. 61/793,007, filed Mar. 15, 2013; U.S. Provisional Application No. 61/882,936, filed Sep. 26, 2013; and U.S. Provisional Application No. 61/893,436, filed Oct. 21, 2013. The contents of these applications are incorporated hereby by reference in their entirety.

TECHNICAL FIELD

This disclosure relates to pharmaceutical compositions comprising ceftolozane, pharmaceutical compositions comprising tazobactam and ceftolozane, methods of preparing those compositions, and related methods and uses thereof.

BACKGROUND

Ceftolozane is a cephalosporin antibacterial agent. The antibacterial activity of ceftolozane is believed to result from its interaction with penicillin binding proteins (PBPs) to inhibit the biosynthesis of the bacterial cell wall which acts to stop bacterial replication. Ceftolozane is also referred to as “CXA-101”, FR264205, (6R,7R)-3-[(5-amino-4-{[(2-aminoethyl)carbamoyl]amino}-1-methyl-1H-pyrazol-2-ium-2-yl)methyl]-7-({(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(1-carboxy-1-methylethoxy)imino]acetyl}amino)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate, or (6R,7R)-3-[5-Amino-4-[3-(2-aminoethyl)ureido]-1-methyl-1H-pyrazol-2-ium-2-ylmethyl]-7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(Z)-1-carboxy-1-methylethoxyimino]acetamido]-3-cephem-4-carboxylic acid). As used herein, the term “ceftolozane” means (6R,7R)-3-[(5-amino-4-{[(2-aminoethyl)carbamoyl]amino}-1-methyl-1H-pyrazol-2-ium-2-yl)methyl]-7-({(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(1-carboxy-1-methylethoxy)imino]acetyl}amino)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate or (6R,7R)-3-[5-Amino-4-[3-(2-aminoethyl)ureido]-1-methyl-1H-pyrazol-2-ium-2-ylmethyl]-7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(Z)-1-carboxy-1-methylethoxyimino]acetamido]-3-cephem-4-carboxylic acid in its free-base or salt form, including a sulfate form. Unless otherwise indicated, the term“CXA-101” as used herein can refer to ceftolozane in any pharmaceutically acceptable form, e.g., ceftolozane in its free-base or salt form, including a ceftolozane sulfate salt form. Ceftolozane sulfate is a pharmaceutically acceptable salt of ceftolozane that can be combined with sodium chloride and other components to obtain an antibiotic composition suitable for administration by injection or infusion.

Antibacterial pharmaceutical compositions can include ceftolozane as a pharmaceutically acceptable salt formulated for intravenous administration. Ceftolozane sulfate is a pharmaceutically acceptable ceftolozane salt of formula (I) that can be formulated for intravenous administration or infusion.

U.S. Pat. No. 7,129,232 discloses ceftolozane and various ceftolozane salts. For example, a ceftolozane hydrogen sulfate salt is disclosed among ceftolozane salts that can be formed “with a base or an acid addition salt such as a salt with an inorganic base, for example, an alkali metal salt [e.g., sodium salt, potassium salt, etc.], an alkaline earth metal salt [e.g., calcium salt, magnesium salt, etc.], an ammonium salt; a salt with an organic base, for example, an organic amine salt [e.g., trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, N,N′-dibenzylethylenediamine salt, etc.]; an inorganic acid addition salt [e.g., hydrochloride, hydrobromide, sulfate, hydrogen sulfate, phosphate, etc.]; an organic carboxylic or sulfonic acid addition salt [e.g., formate, acetate, trifluoroacetate, maleate, tartrate, citrate, fumarate, methanesulfonate, benzenesulfonate, toluenesulfonate, etc.]; and a salt with a basic or acidic amino acid [e.g., arginine, aspartic acid, glutamic acid, etc.].”

Antibiotic pharmaceutical compositions comprising a beta-lactam antibiotic compound (e.g., a cephalosporin) (i.e., an antibiotic compound possessing one or more beta-lactam moieties) can be administered with a beta-lactamase inhibitor (BLI) compound. For example, beta-lactam antibiotic compounds such as ceftolozane or other cephalosporin antibiotic compounds can be formulated with, and/or administered in combination with beta-lactamase inhibiting compounds (e.g., tazobactam and salts thereof) in order to mitigate the effects of bacterial beta-lactamase enzymes that can lead to bacterial resistance to antibiotic therapy. Tazobactam is a BLI compound approved for use in fixed dose combination with piperacillin in an injectable antibacterial product available under commercial names ZOSYN (U.S.) and TAZOCIN (e.g., in Canada, and the United Kingdom). Tazobactam sodium, a derivative of the penicillin nucleus, is a penicillanic acid sulfone having the chemical name sodium (2S,3S,5R)-3-methyl-7-oxo-3-(1H-1,2,3-triazol-1-ylmethyl)-4-thia-lazabicyclo[3.2.0]heptane-2-carboxylate-4,4-dioxide. The chemical formula is C₁₀H₁₁N₄NaO₅S and the molecular weight is 322.3. The chemical structure of tazobactam sodium is:

Ceftolozane can be formulated with tazobactam in antibiotic compositions called CXA-201 (ceftolozane/tazobactam for injection), comprising ceftolozane and tazobactam in a 2:1 weight ratio between the amount of ceftolozane active and the amount of tazobactam acid, regardless of the salt forms of these compositions (e.g., 1,000 mg of ceftolozane active can be included in about 1,147 mg of ceftolozane sulfate). CXA-201 compositions include an amount of tazobactam in a pharmaceutically acceptable form providing 500 mg of tazobactam acid per 1,000 mg of ceftolozane active as acomposition formulated for injection, or for reconstitution prior to parenteral administration. In one product presentation, CXA-201 can be provided in a single container comprising ceftolozane sulfate and tazobactam sodium, administered by reconstituting a container-unit dosage form container of solid CXA-201 to form a reconstituted injectable formulation. In one presentation (e.g., for treatment of certain urinary tract infections and/or certain intr-abdominal infections), each unit dosage form container of CXA-201 can contain 1000 mg of ceftolozane active (free base equivalent weight, e.g., provided as a pharmaceutically acceptable salt such as ceftolozane sulfate) and sterile tazobactam sodium at a quantity equivalent of 500 mg of tazobactam free acid, in a solid form. In another presentation (e.g., for treatment of hospital acquired/ventilator-associated bacterial pneumonia (HABP/VABP)), a CXA-201 product can include a unit dosage form container providing 2,000 mg of ceftolozane active (e.g., as an equivalent amount of ceftolozane sulfate) and 1,000 mg of tazobactam acid (e.g., as an equivalent amount of tazobactam sodium). CXA-201 compositions display potent antibacterial activity against various gram-negative infections such as, for example, complicated intra-abdominal infection (cIAI), complicated urinary tract infection (cUTI), or hospital acquired/ventilator-associated bacterial pneumonia (HABP/VABP).

As disclosed herein, ceftolozane was initially found to be chemically unstable in certain lyophilized compositions evaluated during the development of CXA-101 and CXA-201 pharmaceutical compositions. For example, ceftolozane had a residual rate of about 51% in the absence of a stabilizing agent during both a 3 day stability test at 70 degrees C., indicating loss of almost half of the ceftolozane during the test (Example 2, Table 2 control sample), and a 5.88% reduction in ceftolozane purity during a 7 day stability test at 60 degrees C. in the absence of a stabilizing agent (Example 2, Table 2a control sample). Second, the formation of a number of additional ceftolozane degradation products formed during the preparation of initial compositions was observed by additional peaks using high performance liquid chromatography (HPLC) during stability tests of ceftolozane alone (e.g., Peak P12 in Table 4 of Example 3, and the RT63 peak in Table 15 of Example 8), and testing of compositions with tazobactam and ceftolozane formed by co-lyophilization of ceftolozane and tazobactam (e.g., RRT1.22 peak in Tables 12 and 13 of Example 7). Accordingly, there remains an unmet need to identify formulations and manufacturing methods that effectively stabilize ceftolozane both in a solid and liquid form to provide suitably stable pharmaceutical compositions comprising ceftolozane and tazobactam (both in a powder form for reconstitution and in a reconstituted form for parenteral delivery). These formulations should address the need to provide pharmaceutical compositions having desired levels of ceftolozane and tazobactam potency, as well as levels of impurities that are therapeutically acceptable for parenteral administration.

SUMMARY

As provided herein, ceftolozane can be stabilized in pharmaceutical composition comprising ceftolozane and a stabilizing effective amount of a stabilizing agent selected from the group consisting of: sodium chloride, dextran 40, lactose, maltose, trehalose and sucrose. The pharmaceutical compositions provided herein are based in part on the surprising discovery that ceftolozane pharmaceutical compositions comprising these stabilizing agents demonstrate improved ceftolozane residual rates (e.g., % ceftolozane remaining after 3 days at 70° C. as measured by HPLC) and/or chemical stability (e.g., lower reduction in ceftolozane purity measured by HPLC after 7 days at 60° C. in a stability test) compared control samples comprising ceftolozane without a stabilizing agent.

Accordingly, preferred pharmaceutical antibiotic compositions can include ceftolozane sulfate and a stabilizing agent (e.g., 300 to 500 mg of a stabilizing agent per 1,000 mg ceftolozane active) in a lyophilized unit dosage form (e.g., powder in a container). The unit dosage form can be dissolved with a pharmaceutically acceptable carrier (e.g., 0.9% sodium chloride aqueous isotonic saline and/or water for injection), and then intravenously administered. In certain ceftolozane compositions, the stabilizing agent can be selected from the group consisting of: sodium chloride, lactose, maltose and dextran 40, and/or selected from the group consisting of: sodium chloride, trehalose and sucrose.

In addition, the present disclosure provides ceftolozane pharmaceutical compositions based in part on the surprising discovery that ceftolozane pharmaceutical compositions comprising about 1000 mg of ceftolozane active per 189 mg sodium from sodium chloride demonstrate improved chemical stability and purity compared with pharmaceutical compositions comprising ceftolozane with comparatively less sodium chloride. For example, the invention is based in part on the discovery of the absence of the “RT63 Impurity” (also referred to herein as “Formula III”) in HPLC analysis of pharmaceutical compositions comprising about 1,000 mg of ceftolozane and 189 mg sodium from sodium chloride. By comparison, reducing the amount of sodium chloride relative to ceftolozane in tested compositions resulted in at least 1.5-fold greater impurity at RT=63 minutes (observed by HPLC using a Develosil column ODS-UG-5; 5 micrometers; 250×4.6 mm, a mobile phase of sodium perchlorate buffer solution (pH 2.5)/CH₃CN 90:10 (v/v) at a 1.0 mL/min flow rate and oven temperature of 45° C.). The ceftolozane formulations with reduced levels of sodium were not as stable as the ceftolozane formulation containing about 1,000 mg of ceftolozane per 189 mg sodium from sodium chloride per. Ceftolozane formulations containing about 1,000 mg of ceftolozane effective per stabilizing-effective amount of sodium from sodium chloride maintained the level of RT63 Impurity below the detection limit (e.g., 0.03%) measured by HPLC using a Develosil column ODS-UG-5; 5 micrometers; 250×4.6 mm, a mobile phase of sodium perchlorate buffer solution (pH 2.5)/CH₃CN 90:10 (v/v) at a 1.0 mL/min flow rate and oven temperature of 45° C.

In a further embodiment provided herein, ceftolozane sulfate is stabilized in pharmaceutical compositions by incorporation of an effective amount of an inorganic salt stabilizing agent, in particular 125 to 500 mg (e.g., 480 to 500 mg) of sodium chloride per gram of ceftolozane active. This is based in part on the surprising discovery that ceftolozane pharmaceutical compositions comprising 125 to 500 mg (e.g., 480 to 500 mg) of sodium chloride per 1000 mg of ceftolozane active demonstrate improved ceftolozane purity and chemical stability compared to pharmaceutical compositions comprising ceftolozane with comparatively less sodium chloride. For example, the disclosed pharmaceutical compositions have an improved stability as a decrease in the rate of ceftolozane purity and/or a decrease in the rate of formation of substances characterized by HPLC peaks 1 and 7 identified during a 7-day stability study in Example 5. The disclosed ceftolozane pharmaceutical compositions comprise a stabilizing amount of sodium chloride (e.g., 125 to 500 mg of sodium chloride [more specifically, 480 to 500 mg] per 1000 mg of ceftolozane active). Certain preferred compositions demonstrate improved ceftolozane purity (e.g., Table 6) and chemical stability (e.g., with respect to the composition of HPLC peak 1 in Table 7) compared with pharmaceutical compositions comprising ceftolozane with comparatively less sodium chloride. For example, the disclosed pharmaceutical compositions typically comprise less than about 4% total impurity after being stored for seven days at 60° C., as determined by HPLC using a Develosil column ODS-UG-5; 5 micrometers; 250×4.6 mm, a mobile phase of sodium perchlorate buffer solution (pH 2.5)/CH₃CN 90:10 (v/v) at a 1.0 mL/min flow rate and oven temperature of 45° C. Alternatively, the disclosed pharmaceutical compositions comprise less than about 2% of the impurity represented by Peak 1 after being stored for seven days at 60° C., as determined by HPLC using a Develosil column ODS-UG-5; 5 micrometers; 250×4.6 mm, a mobile phase of sodium perchlorate buffer solution (pH 2.5)/CH₃CN 90:10 (v/v) at a 1.0 mL/min flow rate and oven temperature of 45° C., where Peak 1 has a retention time relative to ceftolozane of 0.1.

In an embodiment, pharmaceutical antibiotic compositions provided herein can include ceftolozane sulfate and stabilizing amount of sodium chloride (e.g., 125 to 500 mg more specifically 480 to 500 mg of sodium chloride and 1,000 mg ceftolozane active) in a unit dosage form (e.g., powder in a container). The unit dosage form can be dissolved with a pharmaceutically acceptable carrier, and then intravenously administered.

In another aspect, provided herein is a pharmaceutical composition comprising 125 mg to 500 mg sodium chloride per 1,000 mg of ceftolozane active, wherein the decrease in ceftolozane total purity is not greater than about 4% after storing the pharmaceutical composition for seven days in a sealed container at 60° C., as determined by HPLC using a Develosil column ODS-UG-5; 5 micrometers; 250×4.6 mm, a mobile phase of sodium perchlorate buffer solution (pH 2.5)/CH₃CN 90:10 (v/v) at a 1.0 mL/min flow rate and oven temperature of 45° C.

In another aspect, provided herein is a pharmaceutical composition comprising 125 mg to 500 mg sodium chloride per 1,000 mg of ceftolozane active, wherein the increase in the amount of the impurity represented by Peak 1 is not greater than about 2% after storing the pharmaceutical composition for seven days at 60° C., as determined by HPLC using a Develosil column ODS-UG-5; 5 micrometers; 250×4.6 mm, a mobile phase of sodium perchlorate buffer solution (pH 2.5)/CH₃CN 90:10 (v/v) at a 1.0 mL/min flow rate and oven temperature of 45° C., where Peak 1 has a retention time relative to ceftolozane of about 0.1.

In embodiments of these aspects, the pharmaceutical composition further comprises L-arginine, or citric acid. In other embodiments, the pharmaceutical composition is formulated for parenteral administration. In another embodiment, the compositions can be in a unit dosage form comprising 125 mg to 500 mg sodium chloride, 1,000 mg of ceftolozane in the form of ceftolozane sulfate, L-arginine and citric acid.

In other embodiments of these aspects, the pharmaceutical composition is lyophilized. In another embodiment, the ceftolozane is ceftolozane sulfate.

In another aspect, provided herein is a unit dosage form injectable pharmaceutical composition comprising 125 mg to 500 mg sodium chloride and 1,000 mg of ceftolozane active present as a composition of formula (I)

In another aspect, provided herein is a pharmaceutical composition comprising 125 mg to 500 mg sodium chloride per 1,000 mg of ceftolozane active present as ceftolozane sulfate, wherein the ceftolozane total purity is at least about 94% after storing the pharmaceutical composition for three days at 60° C., as determined by HPLC using a Develosil column ODS-UG-5; 5 micrometers; 250×4.6 mm, a mobile phase of sodium perchlorate buffer solution (pH 2.5)/CH₃CN 90:10 (v/v) at a 1.0 mL/min flow rate and oven temperature of 45° C.

Applicants have further discovered pharmaceutical compositions comprising ceftolozane and tazobactam with reduced and even undetectable amounts of the compound RRT 1.22, and methods of manufacturing these compositions. This is based in part on the discovery that the formation of RRT 1.22 can be reduced if not completely suppressed by lyophilizing ceftolozane in the absence of tazobactam and then blending the lyophilized ceftolozane with a dry tazobactam composition, such as a tazobactam composition lyophilized in the absence of ceftolozane (See Example 10 and the results reported in Tables 23 and 24). Based on these results, pharmaceutical compositions comprising ceftolozane and tazobactam, and pharmaceutical compositions prepared using ceftolozane and tazobactam are provided herein. In particular, these pharmaceutical compositions can include ceftolozane and/or tazobactam with reduced or even undetectable amounts of the compound RRT 1.22:

In one embodiment, a pharmaceutical composition can include ceftolozane and tazobactam with less than 0.15%, 0.10%, 0.05% or 0.03% by weight; or from 0.03-0.05%, 0.03-0.1% or 0.03-0.15% by HPLC or even undectable amounts of RRT 1.22 (e.g., less than about 0.03% of the compound RRT 1.22 measured by HPLC). These pharmaceutical compositions can be obtained by a process comprising the steps of (a) lyophilizing ceftolozane in the absence of tazobactam to obtain a lyophilized ceftolozane composition; and (b) combining the lyophilized ceftolozane with tazobactam under conditions suitable to obtain said pharmaceutical composition with the aforementioned purity levels. The combination of the lyophilized ceftolozane composition with tazobactam can include blending the lyophilized ceftolozane composition with lyophilized or crystalline tazobactam material.

Also provided herein is a pharmaceutical composition comprising a blend of separately lyophilized tazobactam and ceftolozane sulfate in an amount providing 1,000 mg of ceftolozane active per 500 mg of tazobactam active, further comprising less than 0.15%, 0.10%, 0.05% or 0.03% by weight; from 0.03-0.05%, 0.03-0.1% or 0.03-0.15% by HPLC; or even undectable amounts (e.g., less than about 0.03% by HPLC) of a compound of formula (III) detectable at a retention time relative to ceftolozane of 1.22 by high performance liquid chromatography (HPLC) using a Develosil column ODS-UG-5; 5 micrometers; 250 x 4.6 mm, a mobile phase of sodium perchlorate buffer solution (pH 2.5)/CH₃CN 90:10 (v/v) at a 1.0 mL/min flow rate and oven temperature of 45° C. (hereinafter referred to as the “method of Example 1”).

CXA-201 compositions comprising less than about 0.15%, 0.10%, 0.05% or 0.03% by weight; or from 0.03-0.05%, 0.03-0.1% or 0.03-0.15% by HPLC of the compound of formula (III) can be obtained by a process comprising the steps of: (a) forming a first aqueous solution comprising ceftolozane (e.g., in a pharmaceutically acceptable salt such as formula (I)), (b) lyophilizing the first aqueous solution to obtain a lyophilized ceftolozane composition, and (c) blending the lyophilized ceftolozane composition with a tazobactam composition (e.g., tazobactam acid lyophilized in the absence of ceftolozane) in an amount that provides a 2:1 weight ratio between the amount of ceftolozane active and tazobactam active.

In yet another aspect, provided herein is a method for the treatment of a bacterial infection in a mammal, comprising administering to said mammal a therapeutically effective amount of any one of the pharmaceutical compositions provided herein. In an embodiment, the bacterial infection is caused by the bacterial infection is caused by bacteria selected from the group consisting of: Staphylococcus aureus, Escherichia coli, Acinetobacter baumanii, Haemophilus influenzae, Klebsiella pneumonia, and Pseudomonas aeruginosa. In another embodiment, the bacterial infection is selected from the group consisting of nosocomial pneumonia, complicated intra-abdominal infection and complicated urinary tract infection.

In yet another aspect, any of the pharmaceutical compositions provided herein may be used for the manufacture of a medicament for the treatment of complicated intra-abdominal infection (cIAI), complicated urinary tract infection (cUTI), or hospital acquired/ventilator-associated bacterial pneumonia (HABP/VABP).

In still another aspect provided herein, an antibiotic pharmaceutical composition comprises ceftolozane (or a pharmaceutically acceptable salt thereof) and tazobactam (or a pharmaceutically acceptable salt thereof) in a fixed dose combination of 1,000 mg of ceftolozane active per 500 mg of tazobactam active, and a ceftolozane-stabilizing amount of 125 mg to 500 mg sodium chloride per 1,000 mg of ceftolozane active.

In a further aspect disclosed herein, a pharmaceutical composition comprising stabilized ceftolozane sulfate is obtained by a process comprising lyophilizing an aqueous solution comprising 125 mg to 500 mg sodium chloride with an amount of ceftolozane sulfate providing 1,000 mg of ceftolozane active, to obtain the lyophilized stabilized ceftolozane sulfate composition.

Yet another aspect provided herein discloses an antibacterial pharmaceutical composition comprising ceftolozane sulfate and tazobactam in a ratio of 1,000 mg ceftolozane active per 500 mg of tazobactam active, the pharmaceutical composition obtained by a process comprising the steps of:

a) lyophilizing a first aqueous solution in the absence of tazobactam, the first aqueous solution comprising ceftolozane sulfate prior to lyophilization to obtain a first lyophilized ceftolozane composition; and

b) blending the first lyophilized ceftolozane composition with tazobactam to obtain an antibacterial composition comprising less than 0.13% by HPLC of a compound of formula (III) detectable at a retention time relative to ceftolozane of 1.22 by high performance liquid chromatography using a Develosil column ODS-UG-5; 5 micrometers; 250×4.6 mm, a mobile phase of sodium perchlorate buffer solution (pH 2.5)/CH₃CN 90:10 (v/v) at a 1.0 mL/min flow rate and oven temperature of 45° C.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a flowchart showing the steps for preparing a CXA-201 composition comprising ceftolozane (referred to as CXA-101) and tazobactam using a blending process, wherein the ceftolozane and tazobactam are lyophilized separately prior to blending as described herein.

FIG. 2 is a flowchart showing the steps for preparing a CXA-201 composition comprising ceftolozane (referred to as CXA-101) and tazobactam using a co-lyophilization process, as described herein.

FIG. 3 is a reference HPLC chromatogram showing the peaks of ceftolozane (CXA-101) and related composition peaks.

FIG. 4 is a plot of the data points from Table 6, showing the purity of the ceftolozane in CXA-101 compositions at 60° C. on day 0, day 1, day 3, and day 7, as measured by HPLC, wherein the CXA-101 compositions comprise ceftolozane and sodium chloride.

FIG. 5 is a plot of the data points from Table 7, showing the peak area of the composition peak 1 in CXA-101 compositions at 60° C. on day 0, day 1, day 3, and day 7, as measured by HPLC, wherein the CXA-101 compositions comprise ceftolozane and sodium chloride.

FIG. 6 is a plot of the data points from Table 8, showing the total peak area of the composition with a RRT of 0.43 and the composition peak 3 in CXA-101 compositions at 60° C. on day 0, day 1, day 3, and day 7, as measured by HPLC, wherein the CXA-101 compositions comprise ceftolozane and sodium chloride.

FIG. 7 is a plot of the data points from Table 9, showing the peak area of the composition peak 7 in CXA-101 compositions at 60° C. on day 0, day 1, day 3, and day 7, as measured by HPLC, wherein the CXA-101 compositions comprise ceftolozane and sodium chloride.

FIG. 8 is a plot of the data points from Table 17, showing the purity of ceftolozane in CXA-201 compositions at 60° C. on day 0, day 1, day 3, and day 7, as measured by HPLC, wherein the CXA-201 compositions comprise ceftolozane, tazobactam, and sodium chloride.

FIG. 9 is a plot of the data points from Table 18, showing the peak area of the composition peak 1 in CXA-201 compositions at 60° C. on day 0, day 1, day 3, and day 7, as measured by HPLC, wherein the CXA-201 compositions comprise ceftolozane, tazobactam, and sodium chloride.

FIG. 10 is a plot of the data points from Table 19, showing the total peak area of the composition with a RRT of 0.43 and the composition peak 3 in CXA-201 compositions at 60° C. on day 0, day 1, day 3, and day 7, as measured by HPLC, wherein the CXA-201 compositions comprise ceftolozane, tazobactam, and sodium chloride.

FIG. 11 is a plot of the data points from Table 20, showing the peak area of the composition peak 7 in CXA-201 compositions at 60° C. on day 0, day 1, day 3, and day 7, as measured by HPLC, wherein the CXA-201 compositions comprise ceftolozane, tazobactam, and sodium chloride.

FIG. 12 is a flowchart showing the manufacturing process for a ceftolozane/tazobactam composition via co-filling.

FIG. 13a is a flowchart showing the process for preparing a CXA-201 composition comprising ceftolozane (referred to as CXA-101) and tazobactam using a blending process in a dedicated production area according to FDA Guidance.

FIG. 13b is a flowchart showing the process for preparing a ceftolozane/tazobactam composition via co-filling in a dedicated production area according to FDA Guidance.

FIG. 14 shows the mass spectra obtained for the RRT 1.22 compound.

FIG. 15 shows the chemical structures for certain peaks in the spectra in FIG. 14.

DETAILED DESCRIPTION I. Stabilizing Ceftolozane

Ceftolozane can be stabilized in a pharmaceutical composition comprising ceftolozane and a stabilizing effective amount of a stabilizing agent selected from the group consisting of: sodium chloride, dextran 40, lactose, maltose, trehalose and sucrose. The stabilizing agent and the stabilizing effective amount of the stabilizing agent for combination with ceftolozane were determined by high performance liquid chromatography (HPLC) analysis, for example by detecting the ratio of peak areas obtained for ceftolozane compared to peaks for other substances.

Preferred stabilized ceftolozane compositions have a ceftolozane residual rate of greater than the residual rate measured for a comparable ceftolozane composition without the stabilizing agent. Unless otherwise indicated, the residual rate is measured by detecting the amount of ceftolozane in a sample before and after a stability test using HPLC, and determining the percentage of ceftolozane last during the stability test.

Referring to Example 2 (including Table 2), the residual rate of ceftolozane in the control sample without a stabilizing agent (i.e., 100 mg of ceftolozane) after 3 days at 70 degrees C. was 51.2%, meaning that the HPLC peak area after the stability test for ceftolozane was about 51.2% of the HPLC peak area for ceftolozane at the start of the stability test (i.e., 3 days at 70 degrees C.). Sodium chloride, dextran 40, lactose and maltose all showed higher ceftolozane residual rates than the control in Example 2, while ceftolozane was less stable than the control when combined with fructose, xylitol, sorbitol and glucose (e.g., as evidenced by a residual rate lower than that of the control). In one embodiment, stabilized ceftolozane compositions comprise ceftolozane (e.g., ceftolozane sulfate) and a stabilizing effective amount of a stabilizing agent selected from the group consisting of: sodium chloride, dextran 40, lactose and maltose, where the stabilizing effective amount provides a residual rate of at least 51.2% for the ceftolozane in the stabilized ceftolozane composition after 3 days at 70 degrees C. Preferably, the stabilized ceftolozane pharmaceutical compositions after 3 days at 70 degrees C. can comprise at least about 70% of an initial amount of the stabilized ceftolozane in the pharmaceutical composition (i.e., a residual rate of about 70% or greater, as shown in Example 2), where the % of ceftolozane is measured by high performance liquid chromatography (HPLC) according to Example 1.

Referring to Example 2 (Table 2a), stabilized ceftolozane compositions are characterized by a reduction in ceftolozane of less than about 5% after 7 days at 60 degrees C., where the % reduction of ceftolozane is measured by HPLC according to Example 1. The stabilized ceftolozane pharmaceutical composition comprising ceftolozane and a stabilizing agent selected from the group consisting of: sodium chloride, trehalose and sucrose can lose less than 5% of the amount of ceftolozane after 7 days at 60 degrees C., where the % loss of ceftolozane is measured by HPLC according to Example 1. Sodium chloride, trehalose and sucrose all showed reduced reductions in ceftolozane purity after a 7 day stability test at 60 degrees C. (as measured by the % HPLC peak corresponding to ceftolozane before and after the stability test). In one embodiment, stabilized ceftolozane compositions comprise ceftolozane (e.g., ceftolozane sulfate) and a stabilizing effective amount of a stabilizing agent selected from the group consisting of: sodium chloride, trehalose and sucrose, where the stabilizing effective amount provides a reduction in ceftolozane purity of not more than about 5% (e.g, not more than about 4%) for the ceftolozane in the stabilized ceftolozane composition after 3 days at 70 degrees C.

Accordingly, in one aspect, provided herein is a pharmaceutical composition comprising stabilized ceftolozane obtained by a process comprising lyophilizing a composition including ceftolozane and a stabilizing agent selected from the group consisting of: sodium chloride, dextran 40, lactose, maltose, tehalose and sucrose, to obtain a lyophilized stabilized ceftolozane pharmaceutical composition. In an embodiment, the stabilizing agent is selected from the group consisting of: sodium chloride, trehalose and sucrose. In another aspect, provided herein is a pharmaceutical composition comprising stabilized ceftolozane and a stabilizing agent selected from the group consisting of: sodium chloride, dextran 40, lactose, maltose, tehalose and sucrose, wherein the pharmaceutical composition after 3 days at 70 degrees C. comprises at least about 70% of an initial amount of the stabilized ceftolozane in the pharmaceutical composition.

In another aspect, provided herein is a container containing a unit dosage form of a pharmaceutical composition formulated for parenteral administration for the treatment of complicated intra-abdominal infections or complicated urinary tract infections, the pharmaceutical composition comprising 1,000 mg of ceftolozane active, L-arginine, citric acid and about 300-500 mg of a stabilizing agent selected from the group consisting of: sodium chloride, trehalose, and sucrose, wherein the pharmaceutical composition after 3 days at 70 degrees C. comprises at least about 70% of an initial amount of the ceftolozane active in the pharmaceutical composition.

Various ceftolozane compositions are described herein. One stabilized ceftolozane composition comprises ceftolozane (e.g., ceftolozane sulfate), L-arginine, citric acid, and a stabilizing agent. Preferably, the stabilized ceftolozane composition comprises 1,000 mg of ceftolozane active, L-arginine and stabilizing-effective amount of the stabilizing agent. The stabilizing effective amount can be readily determined using HPLC and a stability test as disclosed herein. The stabilizing-effective amount can be effective to provide: (1) a residual rate measured by HPLC of ceftolozane of at least about 51.2% (including, e.g., at least about 70%, and at least about 80%) after 3 days at 70 degrees C. and/or (2) a reduction in ceftolozane purity measured by HPLC of not more than about 5.11% (including, e.g., reductions of not more than about 5%, or 4%) after 7 days at 60 degrees C. Examples of stabilizing effective amounts include 100 mg-500 mg of the stabilizing agent per 1,000 mg of the ceftolozane active, more preferably about 300-500 mg of the stabilizing agent per 1,000 mg of the ceftolozane active.

In the screening of ceftolozane stabilizing agents, it has been found that, surprisingly, a preferred amount of sodium chloride can improve the stability of ceftolozane, including ceftolozane in the ceftolozane sulfate form. For example, in one experiment, a ceftolozane composition comprising about 100 mg (about 1.71 mmol) sodium chloride per 100 mg (about 0.15 mmol) of ceftolozane was more stable compared to many ceftolozane compositions comprising known stabilizing sugars, such as fructose, xylitol, sorbitol, glucose, and D-mannitol, and as stable as other ceftolozane compositions comprising the same amount of certain sugars, such as dextran 40, lactose, and maltose (see Example 2). Interestingly, additional experiments demonstrated that the use of maltose in a ceftolozane composition resulted in a significant amount of additional compounds (see Example 3).

Surprisingly, pharmaceutical compositions comprising ceftolozane and 125 to 1000 mg sodium chloride per 1000 mg of ceftolozane have been observed to exhibit better chemical stability over the course of time and/or in the presence of heat, and fewer additional compounds than those pharmaceutical compositions comprising ceftolozane and less sodium chloride (i.e., less than 125 mg sodium chloride per 1000 mg of ceftolozane) (see, e.g., Example 5). In particular embodiments described herein, the pharmaceutical compositions comprising ceftolozane and 125 to 500 mg sodium chloride per 1000 mg of ceftolozane have been found to be more stable than the compositions comprising ceftolozane and less than 125 mg sodium chloride per 1000 mg of ceftolozane.

Ceftolozane compositions having 50-481 mg of sodium chloride per 1,000 mg ceftolozane active were prepared as described in Table 5 and tested for stability as described in Example 5. Ceftolozane was more stable in compositions containing at least 125 mg of sodium chloride per 1,000 mg of ceftolozane active, as measured by high performance liquid chromatography (HPLC) analysis by detecting the ratio of peak areas obtained for ceftolozane compared to peaks for other substances. (Unless otherwise indicated, HPLC measurements reported herein are obtained using a Develosil column ODS-UG-S; 5 micrometers; 250×4.6 mm, a mobile phase of sodium perchlorate buffer solution (pH 2.5)/CH₃CN 90:10 (v/v) at a 1.0 mL/min flow rate and oven temperature of 45° C.) During the stability test of Example 5, ceftolozane samples containing 125 mg, 190 mg and 481 mg of sodium chloride per 1,000 mg of ceftolozane active showed a decrease in ceftolozane total purity measured by HPLC that was at least about 35% less than reductions in ceftolozane total purity observed for formulations containing 50 mg or 75 mg sodium chloride per 1,000 mg ceftolozane active. Thus, ceftolozane compositions having at least 125 mg or more sodium chloride relative to the fixed amount of ceftolozane were about 35-90% more stable than comparable ceftolozane compositions having less than 125 mg sodium chloride (e.g., the % decrease in ceftolozane for the sample containing 75 mg sodium chloride was about 35% greater than the comparable % decrease in ceftolozane for the sample containing 190 mg sodium chloride). In addition, samples obtained from ceftolozane compositions containing 125 mg, 190 mg and 481 mg of sodium chloride per 1,000 mg of ceftolozane active showed a decrease in ceftolozane that was up to about 90% less than reductions in ceftolozane observed for formulations containing 50 mg or 75 mg sodium chloride per (e.g., the % decrease in ceftolozane for the sample containing 50 mg sodium chloride was about 90% greater than the comparable % decrease in ceftolozane for the sample containing 481 mg sodium chloride).

The ceftolozane sodium-stabilized compositions having 125 mg or more sodium chloride relative to the fixed amount of 1,000 mg ceftolozane active also had lower quantities of additional substances identified by peaks 1 and 7 having characteristic retention times measured by HPLC (see Table 1, indicating retention times of about 0.1 for peak 1 and about 1.3 for peak 7 relative to ceftolozane measured according to the HPLC method of Example 1). In particular, these sodium chloride stabilized ceftolozane compositions were characterized by about 37-94% less of the material of peak 1 and about 38-306% less of the material of peak 7 (measured by corresponding HPLC peak areas) than comparable ceftolozane compositions having less than 125 mg sodium chloride (e.g., see 7-day stability study in Example 5). Referring to the data in Table 7 (FIG. 5), the amount of the composition of peak 1 (measured by HPLC according to Example 1) was measured by the % increase in the peak 1 HPLC peak during the 7-day stability test of Example 5.

In particular, samples containing 125 mg, 190 mg and 481 mg of sodium chloride per 1,000 mg of ceftolozane active showed at least a 37% reduction in the amount of the peak 1 composition observed for these formulations containing at least 125 mg sodium chloride per 1,000 mg ceftolozane active, compared to the compositions with 50 mg or 75 mg sodium chloride per 1,000 mg of ceftolozane active (e.g., the % increase in peak 1 for the sample containing 75 mg sodium chloride was about 37% greater than the comparable % decrease in ceftolozane for the sample containing 190 mg sodium chloride). In addition, compositions containing 125 mg, 190 mg and 481 mg of sodium chloride per 1,000 mg of ceftolozane active showed up to a 94% reduction in the amount of the peak 1 composition observed for these formulations containing at least 125 mg sodium chloride per 1,000 mg ceftolozane active, compared to the compositions with 50 mg or 75 mg sodium chloride per 1,000 mg of ceftolozane active (e.g., the % increase in peak 1 for the sample containing 50 mg sodium chloride was about 94% greater than the comparable % decrease in ceftolozane for the sample containing 481 mg sodium chloride).

The formulation of pharmaceutical compositions can be selected to minimize decomposition of the constituent drug substances and to produce a composition that is stable under a variety of storage conditions.

Provided herein are pharmaceutical compositions useful for the treatment of bacterial infections comprising ceftolozane and sodium chloride, wherein the sodium chloride is present in an amount sufficient to stabilize the ceftolozane. Also provided herein are pharmaceutical compositions comprising ceftolozane, tazobactam, and sodium chloride, wherein the sodium chloride is present in an amount sufficient to stabilize the ceftolozane. Advantageously, these pharmaceutical compositions have fewer additional compounds and are more chemically stable, and can therefore be stored for longer periods of time.

In one embodiment, provided herein is a pharmaceutical composition comprising ceftolozane and 125 mg sodium chloride per 1000 mg of ceftolozane, e.g., 125 to 500 mg sodium chloride per 1000 mg of ceftolozane, 200-500 mg sodium chloride per 1000 mg of ceftolozane, 300-500 mg sodium chloride per 1000 mg of ceftolozane, 400-500 mg sodium chloride per 1000 mg of ceftolozane, 450-500 mg sodium chloride per 1000 mg of ceftolozane, 460-500 mg sodium chloride per 1000 mg of ceftolozane, or about 476 mg sodium chloride per 1000 mg of ceftolozane, wherein the purity of the ceftolozane in the composition is 75% or greater after 3 days at 70° C. In another embodiment, provided herein is a pharmaceutical composition comprising ceftolozane and about 487 mg sodium chloride per 1000 mg of ceftolozane, wherein the purity of the ceftolozane in the composition is 75% or greater after 3 days at 70° C. In certain embodiments, the purity of the ceftolozane in the composition is 80% or greater, 85% or greater, 90% or greater, 95% or greater, 97% or greater, or 99% or greater after 3 days at 70° C.

In another embodiment, provided herein is a pharmaceutical composition comprising ceftolozane and 125 mg sodium chloride per 1000 mg of ceftolozane, e.g., 125 to 500 mg sodium chloride per 1000 mg of ceftolozane, 200-500 mg sodium chloride per 1000 mg of ceftolozane, 300-500 mg sodium chloride per 1000 mg of ceftolozane, 400-500 mg sodium chloride per 1000 mg of ceftolozane, 450-500 mg sodium chloride per 1000 mg of ceftolozane, 460-500 mg sodium chloride per 1000 mg of ceftolozane, or about 476 mg sodium chloride per 1000 mg of ceftolozane, wherein the purity of the ceftolozane in the composition is 94.8% or greater after 3 days at 60° C. In another embodiment, provided herein is a pharmaceutical composition comprising ceftolozane and about 487 mg sodium chloride per 1000 mg of ceftolozane, wherein the purity of the ceftolozane in the composition is 94.8% or greater after 3 days at 60° C. In certain embodiments, the purity of the ceftolozane in the composition is 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater after 3 days at 60° C.

In still another embodiment, provided herein is a pharmaceutical composition comprising ceftolozane and 125 mg sodium chloride per 1000 mg of ceftolozane, e.g., 125 to 500 mg sodium chloride per 1000 mg of ceftolozane, 200-500 mg sodium chloride per 1000 mg of ceftolozane, 300-500 mg sodium chloride per 1000 mg of ceftolozane, 400-500 mg sodium chloride per 1000 mg of ceftolozane, 450-500 mg sodium chloride per 1000 mg of ceftolozane, 460-500 mg sodium chloride per 1000 mg of ceftolozane, or about 476 mg sodium chloride per 1000 mg of ceftolozane, wherein the purity of the ceftolozane in the composition decreases by 3.1% or less after 3 days at 60° C. In another embodiment, provided herein is a pharmaceutical composition comprising ceftolozane and about 487 mg sodium chloride per 1000 mg of ceftolozane, wherein the purity of the ceftolozane in the composition decreases by 3.1% or less after 3 days at 60° C. In certain embodiments, the purity of the ceftolozane in the composition decreases by 3.0% or less, 2.5% or less, 2.0% or less, 1.5% or less, or 1% or less after 3 days at 60° C.

In another aspect, provided herein is a pharmaceutical composition comprising about 1,000 mg of ceftolozane active per 189 mg sodium from sodium chloride, and not more than 0.03% by high performance liquid chromatography (HPLC) of a RT63 Impurity at a retention time of about 63 minutes observed by HPLC using a Develosil column ODS-UG-5; 5 micrometers; 250×4.6 mm, a mobile phase of sodium perchlorate buffer solution (pH 2.5)/CH₃CN 90:10 (v/v) at a 1.0 mL/min flow rate and oven temperature of 45° C., the pharmaceutical composition obtained by a process comprising the step of lyophilizing an aqueous solution comprising 189 mg sodium from sodium chloride per 1,000 mg of ceftolozane active in the form of ceftolozane sulfate to obtain a lyophilized ceftolozane composition, and formulating the pharmaceutical composition from the lyophilized ceftolozane composition.

In one embodiment, the pharmaceutical composition comprises a total of 1,000 mg of ceftolozane active. In another aspect, provided herein is a pharmaceutical composition obtained by a process comprising the step of lyophilizing an aqueous solution comprising 189 mg sodium from sodium chloride per 1,000 mg of ceftolozane in the form of ceftolozane sulfate to obtain a lyophilized ceftolozane composition.

In one embodiment, the pH of the aqueous solution is 5.0 to 7.0, e.g., 6.0 to 7.0, and the aqueous solution further comprises L-arginine. In another embodiment, the pharmaceutical composition is formulated for parenteral administration and further comprises citric acid. In another embodiment, the composition is a unit dosage form in a container comprising tazobactam and 189 mg sodium from sodium chloride per 1,000 mg of ceftolozane active in the form of ceftolozane sulfate.

In another embodiment, the aqueous solution further comprises L-arginine and citric acid;

the pH of the aqueous solution is 6.0 to 7.0 prior to lyophilization; and the pharmaceutical composition further comprises tazobactam blended with the lyophilized ceftolozane composition.

In still another aspect, provided herein is a container containing a unit dosage form of a pharmaceutical composition formulated for parenteral administration for the treatment of complicated intra-abdominal infections or complicated urinary tract infections, the pharmaceutical composition comprising 189 mg sodium from sodium chloride, and 1,000 mg ceftolozane active in the form of ceftolozane sulfate.

In one embodiment, the container comprises the ceftolozane sulfate, tazobactam and the sodium chloride and not more than 0.03% by high performance liquid chromatography (HPLC) of a RT63 Impurity at a retention time of about 63 minutes observed by HPLC using a Develosil column ODS-UG-5; 5 micrometers; 250×4.6 mm, a mobile phase of sodium perchlorate buffer solution (pH 2.5)/CH₃CN 90:10 (v/v) at a 1.0 mL/min flow rate and oven temperature of 45° C.

Typically, antibiotic compositions do not contain sodium chloride or contain only a small amount of sodium chloride. For example, Maxipime®, which is approved for pneumonia, empiric therapy for febrile neutropenic patients, uncomplicated and complicated urinary tract infections, uncomplicated skin and skin structure infections, and complicated intra-abdominal infections, is a dry mixture of cefepime hydrochloride and L-arginine, wherein the mixture does not contain sodium chloride. Cefazolin® for injection, which is approved for respiratory tract infections, urinary tract infections, skin and skin structure infections, biliary tract infections, bone and joint infections, genital infections, septicemia, and endocarditis and perioperative prophylaxis, comprises lyophilized cefazolin sodium that does not contain additional sodium salt. Furthermore, Rocephin®, which is approved for lower respiratory tract infections, acute bacterial otitis media, skin and skin structure infections, urinary tract infections, uncomplicated gonorrhea, pelvic inflammatory disease, bacterial septicemia, bone and joint infections, intra-abdominal infections, meningitis, and surgical prophylaxis, comprises ceftriaxone sodium that only comprises 13.5 mg of free sodium per 1000 mg of ceftriaxone sodium, which equals about 34 mg sodium chloride per 1000 mg of ceftriaxone sodium if the free sodium is in sodium chloride form. In contrast, the pharmaceutical compositions provided herein (compositions comprising ceftolozane and sodium chloride, and compositions comprising ceftolozane, tazobactam, and sodium chloride), have high amounts of sodium chloride, e.g., 125-1000 mg sodium chloride per 1000 mg of ceftolozane.

Ceftolozane

The compound 5-amino-4-{[(2-aminoethyl)carbamoyl]amino}-2-{[(6R,7R)-7-({(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(1-carboxy-1-methylethoxy)imino]acetyl}amino)-2-carboxy-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl]methyl}-1-methyl-1H-pyrazolium monosulfate (also known also as ceftolozane sulfate, FR264205, “CXA-101”) is a cephalosporin compound (shown below), the synthesis of which is described in U.S. Pat. No. 7,129,232, wherein the compound is also named 7β-[(Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-(1-carboxy-1-methylethoxyimino)acetamido]-3-{3-amino-4-[3-(2-aminoethyl)ureido]-2-methyl-1-pyrazolio}methyl-3-cephem-4-carboxylate. Ceftolozane has the chemical formula below and CAS registry number 689293-68-3. “Ceftolozane” can be provided as the salt, ceftolozane sulfate.

Unless otherwise indicated herein, the phrase “1000 mg ceftolozane” or “1 g ceftolozane” refers to an amount of ceftolozane containing the free base equivalent weight of ceftolozane provided in the free base form or any suitable salt form, as appropriate. For example, a composition containing 1000 mg of ceftolozane in the ceftolozane sulfate solid form will include greater than 1000 mg of material (e.g., due to at least the additional weight of the sulfate counter ion). Preferably, the ceftolozane is present as ceftolozane sulfate. If a ceftolozane sulfate composition contains “1000 mg of ceftolozane” then it includes an amount of ceftolozane sulfate comprising 1000 mg of the ceftolozane molecule in free base equivalent form. For example, as shown in Table 29, 1147 mg ceftolozane sulfate corresponds to 1000 mg of ceftolozane free base.

In another embodiment, “1000 mg ceftolozane” refers to an amount of ceftolozane that is considered a bioequivalent by the United States Food and Drug Administration (FDA), i.e. for which 90% CI of the relative mean Cmax, AUC(0-t) and AUC(0-∞) is within 80.00% to 125.00% of the reference formulation in the fasting state (see: “Guidance for Industry: Bioavailability and Bioequivalence Studies for Orally Administered Drug Products—General Considerations”. Center for Drug Evaluation and Research, United States Food and Drug Administration, 2003).

“Ceftolozane active” refers to the active portion of a salt form of ceftolozane, i.e., the free base form of ceftolozane.

As used herein, “125 to 1000 mg sodium chloride per 1000 mg of ceftolozane” refers to a ratio of sodium chloride to ceftolozane free base equivalent. For example, “125 to 1000 mg sodium chloride per 1000 mg of ceftolozane” includes, for example, 62.5 to 500 mg sodium chloride per 500 mg of ceftolozane, as well as, for example, 25 to 200 mg sodium chloride per 200 mg ceftolozane, etc.

In another aspect, provided herein is a method for the treatment of bacterial infections in a mammal, comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising ceftolozane as described herein.

II. Ceftolozane in the Presence of Tazobactam

It has also been observed that pharmaceutical compositions comprising ceftolozane, tazobactam, and 125 to 1000 mg sodium chloride per gram of ceftolozane exhibit better chemical stability and fewer additional compounds than those pharmaceutical compositions comprising ceftolozane and tazobactam, but less sodium chloride (see, e.g., Example 8). In particular embodiments described herein, the pharmaceutical compositions comprising ceftolozane, tazobactam, and 125 to 500 mg sodium chloride per 1000 mg of ceftolozane have been found to be more stable than the compositions comprising ceftolozane, tazobactam, and less than 125 mg sodium chloride per gram of ceftolozane.

Adding high amounts of sodium chloride to CXA-201 compositions (e.g., 125-1000 mg sodium chloride per 1000 mg of ceftolozane, 125-500 mg sodium chloride per 1000 mg of ceftolozane, 200-500 mg sodium chloride per 1000 mg of ceftolozane, 300-500 mg sodium chloride per 1000 mg of ceftolozane, 400-500 mg sodium chloride per 1000 mg of ceftolozane, 450-500 mg sodium chloride per 1000 mg of ceftolozane, 460-500 mg sodium chloride per 1000 mg of ceftolozane, or about 476 mg sodium chloride per 1000 mg of ceftolozane) also inhibits the formation of certain additional compounds. Adding about 487 mg sodium chloride per 1000 mg of ceftolozane to CXA-201 composition can also inhibit the formation of certain additional compounds. For example, in one experiment, CXA-201 compositions comprising 125-481 mg sodium chloride per 1000 mg ceftolozane developed a reduced amount of a composition having a retention time of 63 minutes (“RT 63′”) after three months at 25° C. (see the HPLC measurements shown in Example 8A).

Accordingly, in one aspect, provided herein is a pharmaceutical composition comprising ceftolozane, tazobactam, and 125-1000 mg sodium chloride per 1000 mg of ceftolozane, e.g., 125-500 mg sodium chloride per 1000 mg of ceftolozane, 200-500 mg sodium chloride per 1000 mg of ceftolozane, 300-500 mg sodium chloride per 1000 mg of ceftolozane, 400-500 mg sodium chloride per 1000 mg of ceftolozane, 450-500 mg sodium chloride per 1000 mg of ceftolozane, 460-500 mg sodium chloride per 1000 mg of ceftolozane, or about 476 mg sodium chloride per 1000 mg of ceftolozane. In another embodiment, provided herein is a pharmaceutical composition comprising ceftolozane, tazobactam, and about 487 mg sodium chloride per 1000 mg of ceftolozane. In another embodiment, provided herein is a pharmaceutical composition comprising ceftolozane, tazobactam, and 125-1000 mg sodium chloride per 1000 mg of ceftolozane, e.g., 125-500 mg sodium chloride per 1000 mg of ceftolozane, 200-500 mg sodium chloride per 1000 mg of ceftolozane, 300-500 mg sodium chloride per 1000 mg of ceftolozane, 400-500 mg sodium chloride per 1000 mg of ceftolozane, 450-500 mg sodium chloride per 1000 mg of ceftolozane, 460-500 mg sodium chloride per 1000 mg of ceftolozane, or about 476 mg sodium chloride per 1000 mg of ceftolozane, wherein the purity of the ceftolozane in the composition is 94.9% or greater after 3 days at 60° C. In another embodiment, provided herein is a pharmaceutical composition comprising ceftolozane, tazobactam, and about 487 mg sodium chloride per 1000 mg of ceftolozane, wherein the purity of the ceftolozane in the composition is 94.9% or greater after 3 days at 60° C. In certain embodiments, the purity of the ceftolozane in the composition is 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater after 3 days at 60° C.

Tazobactam

The compound (2S,3S,5R)-3-methyl-7-oxo-3-(1H-1,2,3-triazol-1-ylmethyl)-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid 4,4-dioxide (also known as tazobactam) is a (3-lactamase inhibitor of the following structure:

As used herein, tazobactam can be a free acid, a sodium salt, an arginine salt, or a hydrate or solvate thereof. The phrases “250-750 mg tazobactam”, “250-700 mg tazobactam,” “300-700 mg tazobactam”, “300-650 mg tazobactam”, “350-650 mg tazobactam”, “350-600 mg tazobactam”, “400-600 mg tazobactam”, “400-550 mg tazobactam”, “450-550 mg tazobactam” or “about 500 mg tazobactam” refer to an amount of tazobactam containing the free acid equivalent weight of tazobactam provided in the free acid form or any suitable salt form. For example, a composition containing 500 mg of tazobactam in the tazobactam sodium solid form will include greater than 500 mg of material (e.g., due to at least the additional weight of the sodium counter ion). For example, as shown in Table 29, 537 mg tazobactam sodium corresponds to 500 mg of tazobactam free acid. Preferably, the tazobactam is present as tazobactam sodium. If a tazobactam sodium composition contains “500 mg of tazobactam” then it includes an amount of tazobactam sodium comprising 500 mg of the tazobactam molecule in free acid equivalent form.

In an embodiment, the tazobactam is tazobactam sodium sterile powder. In yet a further embodiment, the tazobactam sodium sterile powder is generated by neutralizing tazobactam acid with sodium bicarbonate followed by lyophilization.

As used herein, the term “tazobactam active” refers to the active portion of a salt form of tazobactam, i.e., tazobactam free acid.

In certain embodiments, the pharmaceutical compositions further comprise tazobactam sodium at a quantity equivalent of 500 mg of tazobactam free acid in a lyophilized powder form per 1000 mg of ceftolozane (anhydrous, free base equivalent).

III. Liquid Pharmaceutical Compositions or Formulations

In another aspect, provided herein is a liquid pharmaceutical composition (e.g., an intravenous infusion solution) comprising ceftolozane and tazobactam, wherein the composition is suitable for intravenous administration. In one embodiment, the composition further comprises 125-1000 mg sodium chloride per 1000 mg of ceftolozane. In another embodiment, the composition further comprises 125-500 mg sodium chloride per 1000 mg of ceftolozane. In an embodiment, the liquid pharmaceutical composition (e.g., an intravenous infusion solution) is prepared by reconstitution of a ceftolozane and tazobactam composition with sterile water and/or normal sterile saline, followed by dilution with sterile water and/or normal sterile saline. In an embodiment, the liquid pharmaceutical composition (e.g., an intravenous infusion solution) is prepared by reconstitution of a ceftolozane and tazobactam composition with normal sterile saline, followed by dilution with normal sterile saline. In another embodiment, the liquid pharmaceutical composition (e.g., an intravenous infusion solution) has an osmolality between about 300 mOsm/kg and 900 mOsm/kg, including injectable formulations with an osmolality of 350-900 mOsm/kg to 350-800 mOsm/kg, 400-500 mOsm/kg and 500-600 mOsm/kg. In a further embodiment, the liquid pharmaceutical composition (e.g., an intravenous infusion solution) comprising 1,000 mg ceftolozane active and 500 mg of tazobactam active (as pharmaceutically acceptable salts thereof) has an osmolality that is between about 400 mOsm/kg and 500 mOsm/kg (e.g., 446-478 mOsm/kg, 440-480 mOsm/kg, 420-490 mOsm/kg). In a further embodiment, the liquid pharmaceutical composition (e.g., an intravenous infusion solution) comprising 2,000 mg ceftolozane active and 1000 mg of tazobactam active (as pharmaceutically acceptable salts thereof) has an osmolality that is between about 500 mOsm/kg and 650 mOsm/kg. In yet a further embodiment, the liquid pharmaceutical composition (e.g., an intravenous infusion solution) has an osmolality that is less than about 600 mOsm/kg (e.g., 290-610 mOsm/kg, 350-605 mOsm/kg, 550-605 mOsm/kg, 589-604 mOsm/kg). In another embodiment, the ceftolozane and tazobactam of the liquid pharmaceutical composition (e.g., an intravenous infusion solution) are controlled to pH 5 to 7. In a further embodiment, the ceftolozane and tazobactam of the liquid pharmaceutical composition (e.g., an intravenous infusion solution) are controlled to about pH 6.

In one embodiment, the methods further comprise reconstituting the lyophilized mixture in an aqueous solvent, such that the resulting solution is suitable for infusion. The mixture can be reconstituted in saline and/or sterile water for injection.

Methods of Preparing Pharmaceutical Compositions Comprising Ceftolozane and Sodium Chloride

Pharmaceutical compositions comprising ceftolozane and stabilizing-effective amount of a stabilizing agent can be obtained by lyophilization. As is known to those skilled in the art, lyophilization is a process of freeze-drying in which water is sublimed from a frozen solution of one or more solutes. Specific methods of lyophilization are described in Remington's Pharmaceutical Sciences, Chapter 84, page 1565, Eighteenth Edition, A. R. Gennaro, (Mack Publishing Co., Easton, Pa., 1990). A pharmaceutical composition comprising ceftolozane can be prepared by adding a stabilizing amount of sodium chloride in a fixed ratio to ceftolozane in an aqueous solution prior to lyophilization, then lyophilizing the solution to obtain a lyophilized composition comprising sodium chloride and ceftolozane.

In particular, the pharmaceutical antibiotic compositions can include stabilized ceftolozane sulfate obtained by a process comprising the steps of lyophilizing an aqueous solution containing ceftolozane and a stabilizing-effective amount of a stabilizing agent, where the stabilizing-effective amount of the stabilizing agent is about 100 to 500 mg (preferably 300-500 mg) of the stabilizing agent per 1,000 mg ceftolozane active in the aqueous solution prior to lyophilization. A therapeutically effective amount of ceftolozane (e.g., ceftolozane sulfate) and a stabilizing-effective amount of the stabilizing agent can dissolved in an aqueous solution that can be lyophilized to obtain a stabilized ceftolozane pharmaceutical composition.

The method can further comprise the steps of: (1) forming a solution comprising sodium chloride and ceftolozane or a salt thereof followed by lyophilizing the solution; and (2) combining the lyophilized ceftolozane with other components (e.g., a β-lactamase inhibitor, such as tazobactam, or a lyophilized β-lactamase inhibitor, such as a lyophilized tazobactam) to obtain the pharmaceutical composition. The resulting pharmaceutical composition can be a powder for reconstitution to obtain an injectable pharmaceutical composition that can be intravenously administered to a patient. In yet a further embodiment, the method comprises adding 189 mg sodium from sodium chloride per 1000 mg of ceftolozane active in an aqueous solution, then lyophilizing the solution to obtain a lyophilized material comprising sodium chloride and ceftolozane sulfate in a ratio effective to provide a product with less than 0.03% of the RT63 Impurity as detected by the HPLC method of Example 1.

A. Blending

In other embodiments, pharmaceutical compositions comprising ceftolozane can be obtained by methods that include the steps of: (1) adding a stabilizing amount of sodium chloride to ceftolozane optionally followed by co-lyophilizing or spray drying the ceftolozane and sodium chloride; and (2) combining the product of step (1) with other components. For example, the product of step (1) can be combined with a β-lactamase inhibitor, such as tazobactam (CAS#: 89786-04-9), avibactam (CAS#1192500-31-4), Sulbactam (CAS#68373-14-8) and/or clavulanate (CAS#58001-44-8). The beta lactamase inhibitor can be included in a crystalline or amorpous form, such as a lyophilized tazobactam or crystalline tazobactam (e.g., U.S. Pat. Nos. 8,476,425 and 5,763,603) to obtain the pharmaceutical composition.

Pharmaceutical compositions comprising ceftolozane and tazobactam with reduced or even undectable levels of the compound of RRT 1.22 (e.g., including levels of RRT 1.22 that are not detectable by HPLC according to Example 1 and/or comprise less than 0.15%, 0.10%, 0.05% or 0.03% by weight; or from 0.03-0.05%, 0.03-0.1% or 0.03-0.15% by HPLC according to Example 1) can be obtained by blending a first composition comprising a therapeutically effective amount of ceftolozane in the absence of tazobactam with a second composition comprising a therapeutically effective amount of tazobactam in the absence of ceftolozane to form a blended pharmaceutical composition.

Without being bound by theory, the compound RRT 1.22 can be formed by a reaction between ceftolozane and formylacetic acid, a by-product of tazobactam as illustrated in Marunaka et al. (Chem. Pharm. Bull. 1988, Vol. 36 (11), pp. 4478-4487.

FIG. 1 is a flowchart showing the steps for preparing a CXA-201 composition comprising ceftolozane (referred to as CXA-101) and tazobactam using a blending process, wherein the ceftolozane and tazobactam are lyophilized separately prior to blending as described herein.

The (first) ceftolozane composition can be prepared in the absence of tazobactam by forming a first aqueous solution comprising ceftolozane sulfate and other components including excipients, stabilizers, pH adjusting additives (e.g., buffers) and the like. Non-limiting examples of these additives include sodium chloride, citric acid and L-arginine. For example, the use of sodium chloride results in greater stability; L-arginine is used to adjust pH of the aqueous solution (e.g., to pH 6-7) and to increase the solubility of ceftolozane; and citric acid is used to prevent discoloration of the product, due to its ability to chelate metal ions. Preferably, the first aqueous solution comprises about 125 mg-500 mg sodium chloride per 1,000 mg of ceftolozane active. The ceftolozane can be included as an amount of ceftolozane sulfate of formula (I) containing at least about 1,000 mg ceftolozane active. The (first) aqueous solution is then lyophilized to form a first lyophilized ceftolozane composition, which is combined with tazobactam, e.g., the lyophilized tazobactam (e.g., lyophilized tazobactam sodium) or crystalline tazobactam.

The (second) tazobactam composition can be prepared in the absence of ceftolozane by forming a second solution comprising tazobactam. The tazobactam can be included in an amount providing about 500 mg of tazobactam active per 1,000 mg ceftolozane active (i.e., a 1:2 weight ratio of tazobactam active to ceftolozane active). Unless otherwise indicated, tazobactam can be a free acid, a sodium salt, an arginine salt, or a hydrate or solvate thereof. In one embodiment, the tazobactam in the (second) tazobactam composition is tazobactam acid and the second composition further comprises sodium bicarbonate or sodium hydroxide. Lyophilizing tazobactam in the presence of sodium bicarbonate or sodium hydroxide forms a lyophilized tazobactam sodium, which can then be further blended with the (first) lyophilized ceftolozane composition.

Pharmaceutical compositions with reduced or undectable amounts of the compound of RRT 1.22 can be obtained by lyophilizing ceftolozane without formylacetic acid and/or tazobactam under conditions that prevent formation of RRT 1.22 (e.g., Example 9). The presence of RRT 1.22 can be detected by HPLC (e.g., Examples 1, 6 and 7). Specific methods of lyophilization are described in Remington's Pharmaceutical Sciences, Chapter 84, page 1565, Eighteenth Edition, A. R. Gennaro, (Mack Publishing Co., Easton, Pa., 1990). The formation of the compound of formula (III) can be avoided by preventing the reaction of ceftolozane and formylacetic acid. In one embodiment, the compound of formula (III) can be suppressed by separately lyophlizing ceftolozane sulfate and tazobactam in separate solutions, and then blending the lyophilized compositions to form a pharmaceutical composition.

In one aspect, antibiotic pharmaceutical compositions comprising ceftolozane and tazobactam with less than about 0.15%, 0.10%, 0.05% or 0.03% by weight; or from 0.03-0.05%, 0.03-0.1% or 0.03-0.15% by HPLC of the compound of formula (III) are obtained by a process comprising the steps of: (a) lyophilizing ceftolozane in the absence of tazobactam to obtain a lyophilized ceftolozane composition, and (b) blending the lyophilized ceftolozane composition with a composition comprising tazobactam under conditions suitable for attaining the aforementioned purity levels, e.g., by blending with crystalline tazobactam or lyophilized tazobactam.

In another aspect, antibiotic pharmaceutical compositions comprising ceftolozane and tazobactam and less than about 0.15%, 0.10%, 0.05% or 0.03% by weight; or from 0.03-0.05%, 0.03-0.1% or 0.03-0.15% by HPLC of the compound of formula (III) are obtained by a process comprising the steps of: (a) lyophilizing tazobactam in the absence of ceftolozane to obtain a lyophilized tazobactam composition, and (b) blending the lyophilized tazobactam composition with a composition comprising ceftolozane (e.g., lyophilized ceftolozane sulfate).

In a third aspect, antibiotic pharmaceutical compositions comprising ceftolozane and tazobactam and less than about 0.15%, 0.10%, 0.05% or 0.03% by weight; or from 0.03-0.05%, 0.03-0.1% or 0.03-0.15% by HPLC of the compound of formula (III) are obtained by a process comprising the steps of: (a) lyophilizing tazobactam in the absence of ceftolozane to obtain a lyophilized tazobactam composition, (b) lyophilizing ceftolozane in the absence of tazobactam to obtain a lyophilized ceftolozane composition, and (c) blending the lyophilized tazobactam composition with the lyophilized ceftolozane composition.

Pharmaceutical compositions comprising the compound of formula (III), ceftolozane and tazobactam can be formulated to treat infections by parenteral administration (including subcutaneous, intramuscular, and intravenous) administration. Pharmaceutical compositions may additionally comprise excipients, stabilizers, pH adjusting additives (e.g., buffers) and the like. Non-limiting examples of these additives include sodium chloride, citric acid and L-arginine. For example, the use of sodium chloride results in greater stability; L-arginine is used to adjust pH and to increase the solubility of ceftolozane; and citric acid is used to prevent discoloration of the product, due to its ability to chelate metal ions. In one particular embodiment, the pharmaceutical compositions described herein are formulated for administration by intravenous injection or infusion.

Other pharmaceutical antibiotic compositions can include ceftolozane sulfate and the compound of formula (III). For example, pharmaceutical compositions comprising 0.13%, 0.15%, 0.30%, 0.38%, 0.74% or 0.97% of the compound of formula (III) are herein. The pharmaceutical antibiotic compositions can be provided in a unit dosage form (e.g., in a container). The unit dosage form can be dissolved with a pharmaceutically acceptable carrier, and then intravenously administered. The unit dosage form comprises 1000 mg of ceftolozane active and 500 mg tazobactam, typically 1000 mg ceftolozane active as ceftolozane sulfate and 500 mg of tazobactam active as tazobactam sodium, argininate or free acid. The unit dosage forms are commonly stored in containers.

In another aspect, provided herein is a unit dosage form of a pharmaceutical composition comprising 1,000 mg ceftolozane and 500 mg tazobactam, the pharmaceutical composition formulated for parenteral administration for the treatment of complicated intra-abdominal infections or complicated urinary tract infections, the pharmaceutical composition comprising ceftolozane sulfate and tazobactam, obtained by a process comprising the steps of: lyophlizing an aqueous solution to obtain a lyophilized ceftolozane composition, wherein the aqueous solution comprises water, ceftolozane sulfate, 125-500 mg sodium chloride per 1,000 mg ceftolozane active in the aqueous solution, an amount of L-arginine to provide a pH of 6-7 in the solution prior to lyophilization; and blending the lyophilized ceftolozane composition with a lyophilized tazobactam composition in an amount providing the ratio of about 500 mg tazobactam free acid per 1,000 mg of ceftolozane active to obtain the unit dosage form.

Another embodiment of the invention is a container containing a unit dosage form of a pharmaceutical composition formulated for parenteral administration for the treatment of complicated intra-abdominal infections or complicated urinary tract infections. The container can be obtained by a process comprising the steps of: a) lyophilizing an aqueous solution comprising 189 mg sodium from sodium chloride per 1000 mg ceftolozane active in the form of ceftolozane sulfate and further comprising citric acid, and L-arginine to obtain a lyophilized ceftolozane composition; and b) filling a sufficient quantity of the lyophilized composition into a container to obtain a unit dosage form comprising 189 mg sodium from sodium chloride and 1,000 mg of ceftolozane active in the form of ceftolozane sulfate. In one aspect, the pH of the aqueous solution is 6.0 to 7.0. In another aspect the pharmaceutical composition is formulated for parenteral administration by reconstituting the pharmaceutical composition in the container (e.g., with 10 mL of diluent such as water for injection or isotonic saline) followed by addition of the reconstituted pharmaceutical composition to a carrier for injection (e.g., about 100 mL of isotonic saline or other pharmaceutically acceptable carrier for intravenous administration). Optionally, the container is also filled with tazobactam (e.g., a lyophilized tazobactam such as tazobactam sodium). In yet another aspect, the pharmaceutical composition is a liquid composition comprising 189 mg sodium from sodium chloride, 1,000 mg of ceftolozane active and tazobactam in an amount providing about 500 mg tazobactam acid equivalent per 1,000 mg of ceftolozane active, formulated for parenteral administration and the pH of the aqueous solution is 6.0 to 7.0.

The pharmaceutical composition in the container can also be a Ceftolozane/Tazobactam for Injection Drug Product, 1000 mg/500 mg. It is presented as a combination of two sterile active powders in a single container intended for reconstitution and intravenous infusion. In an embodiment, the drug product is prepared by converting ceftolozane sulfate to a sterile drug product intermediate (composition) powder with excipients citric acid, sodium chloride and L-arginine. This can be done by lyophilization, as described herein. Tazobactam sodium drug substance can be presented as a sterile powder without any excipients. The tazobactam sodium drug substance can be lyophilized, spray dried or provided as a crystalline material. The drug product is then prepared by aseptically filling the two powders (e.g., the two separately lyophilized drug powders) sequentially into a single container.

In an embodiment, the container of ceftolozane/tazobactam for injection contains approximately 2255 mg ceftolozane sterile composition powder that contains 1147 mg ceftolozane sulfate, which is equivalent to 1000 mg ceftolozane free base, as well as approximately 537 mg tazobactam sodium sterile drug substance, equivalent to 500 mg tazobactam free acid. At the time of administration, the container is reconstituted with 10 mL vehicle, sterile 5% Dextrose Injection USP, Water for Injection or 0.9% Sodium Chloride Injection USP, then the container contents further diluted in an infusion bag of 0.9% Sodium Chloride Injection USP or 5% Dextrose Injection USP, for administration. The constituents are shown in Table 29.

A pharmaceutical composition can include ceftolozane sulfate and tazobactam in an amount providing 1,000 mg of ceftolozane active per 500 mg of tazobactam active, and 0.03% to 0.15% by HPLC of a compound of formula (III) detectable at a retention time relative to ceftolozane of 1.22 by high performance liquid chromatography using a Develosil column ODS-UG-5; 5 micrometers; 250×4.6 mm, a mobile phase of sodium perchlorate buffer solution (pH 2.5)/CH₃CN 90:10 (v/v) at a 1.0 mL/min flow rate and oven temperature of 45° C. Optionally, the pharmaceutical composition can further include 125 mg to 500 mg of sodium chloride per 1,000 mg of ceftolozane active, and L-arginine. The tazobactam in the composition can be tazobactam sodium.

In one embodiment of these methods of preparing, 125-500 mg sodium chloride per 1000 mg of ceftolozane is combined. In another embodiment of these methods of preparing, the amount of the sodium chloride combined is 200-500 mg sodium chloride per 1000 mg of ceftolozane, 300-500 mg sodium chloride per 1000 mg of ceftolozane, 400-500 mg sodium chloride per 1000 mg of ceftolozane, 450-500 mg sodium chloride per 1000 mg of ceftolozane, 460-500 mg sodium chloride per 1000 mg of ceftolozane, or about 476 mg sodium chloride per 1000 mg of ceftolozane. In another embodiment of these methods of preparing, the amount of the sodium chloride combined is about 487 mg sodium chloride per 1000 mg of ceftolozane.

In another embodiment of these methods of preparing, the method further comprises lyophilizing the ceftolozane in the absence of the tazobactam. In yet another embodiment, the method can further comprise lyophilizing the tazobactam in the absence of the ceftolozane.

Accordingly, in one aspect, provided herein is a pharmaceutical composition comprising ceftolozane and tazobactam, wherein the composition comprises less than 0.5%, 0.4% 0.3%, 0.25%, 0.2%, 0.15%, 0.1%, or 0.05% by weight of the compound RRT 1.22. In another aspect, provided herein is a pharmaceutical composition comprising ceftolozane and tazobactam, wherein the composition comprises less than 0.1% by weight of the compound RRT 1.22. In one embodiment, the pharmaceutical composition comprises less than 0.05% by weight of the compound RRT 1.22. In another embodiment, the pharmaceutical composition comprises less than 0.15% by weight of the compound RRT 1.22. In yet another embodiment, the pharmaceutical composition comprises no detectable amount of the compound RRT 1.22 as measured by HPLC.

In contrast, a greater amount of compound RRT 1.22 was found in compositions of ceftolozane and tazobactam, wherein the compositions were formed through co-lyophilization, i.e., the ceftolozane and tazobactam were combined and co-lyophilized together, as opposed to being individually lyophilized and blended together (see, e.g., Examples 7 and 10).

In one aspect, provided herein is an antibacterial pharmaceutical composition comprising ceftolozane sulfate and tazobactam in a ratio of 1,000 mg ceftolozane active per 500 mg of tazobactam active, the pharmaceutical composition obtained by a process comprising the steps of: lyophilizing a first aqueous solution in the absence of tazobactam, the first aqueous solution comprising ceftolozane sulfate prior to lyophilization to obtain a first lyophilized ceftolozane composition; and blending the first lyophilized ceftolozane composition with tazobactam to obtain an antibacterial composition comprising less than 0.13% by HPLC of a compound of formula (III) (compound RRT 1.22) detectable at a retention time relative to ceftolozane of 1.22 by high performance liquid chromatography using a Develosil column ODS-UG-5; 5 micrometers; 250×4.6 mm, a mobile phase of sodium perchlorate buffer solution (pH 2.5)/CH₃CN 90:10 (v/v) at a 1.0 mL/min flow rate and oven temperature of 45° C.

In one embodiment, the antibacterial composition comprises less than 0.03% of the compound of formula (III) detected by HPLC. In another embodiment, the first aqueous solution further comprises L-arginine in an amount effective to adjust the pH of the first aqueous solution to 6-7 prior to lyophilization to obtain a first lyophilized ceftolozane composition.

In another embodiment, the antibacterial pharmaceutical composition is obtained by a process further comprising the steps of: lyophilizing a second solution comprising tazobactam in the absence of ceftolozane to form a second lyophilized tazobactam composition; and blending the first lyophilized ceftolozane composition and the second lyophilized tazobactam composition to obtain the antibacterial composition.

In another embodiment, the tazobactam in the second solution is tazobactam acid, and wherein the tazobactam acid in the second solution is lyophilized in the presence of sodium bicarbonate to form the second lyophilized tazobactam solution.

In another embodiment, the first aqueous solution comprises L-arginine in an amount effective to provide a pH of about 5-7, e.g., 6-7. In another embodiment, the first aqueous solution comprises 125 mg to 500 mg of sodium chloride per 1,000 mg of ceftolozane active.

In another embodiment, the first aqueous solution further comprises citric acid. In another embodiment, the first aqueous solution consists of ceftolozane sulfate, citric acid, sodium chloride, L-arginine, and water.

In another aspect, provided herein is a unit dosage form of a pharmaceutical composition formulated for parenteral administration for the treatment of complicated intra-abdominal infections or complicated urinary tract infections, the pharmaceutical composition comprising ceftolozane sulfate and tazobactam in a ratio of 1,000 mg ceftolozane active per 500 mg of tazobactam active, the pharmaceutical composition obtained by a process comprising the steps of: lyophilizing a first aqueous solution in the absence of tazobactam, the first aqueous solution comprising ceftolozane sulfate, 125 mg to 500 mg of sodium chloride per 1,000 mg of ceftolozane active, at a pH of 6-7 prior to lyophilization to obtain a first lyophilized ceftolozane composition; lyophilizing a second solution comprising tazobactam in the absence of ceftolozane to form a second lyophilized tazobactam composition; and blending the first lyophilized ceftolozane composition and the second lyophilized tazobactam composition to obtain the antibacterial composition.

In another embodiment, the unit dosage form comprises a total of not more than 0.03% by HPLC of a compound of formula (III) detectable at a retention time relative to ceftolozane of 1.22 by high performance liquid chromatography using a Develosil column ODS-UG-5; 5 micrometers; 250×4.6 mm, a mobile phase of sodium perchlorate buffer solution (pH 2.5)/CH₃CN 90:10 (v/v) at a 1.0 mL/min flow rate and oven temperature of 45° C.

In another embodiment, the unit dosage form comprises a total of 1,000 mg of ceftolozane active and a total of 500 mg of tazobactam active.

In another embodiment, the unit dosage form comprises a total of not more than 0.03% by HPLC of a compound of formula (III) detectable at a retention time relative to ceftolozane of 1.22 by high performance liquid chromatography using a Develosil column ODS-UG-5; 5 micrometers; 250×4.6 mm, a mobile phase of sodium perchlorate buffer solution (pH 2.5)/CH₃CN 90:10 (v/v) at a 1.0 mL/min flow rate and oven temperature of 45° C.

In another aspect, provided herein is a compound of formula (III):

or a pharmaceutically acceptable salt thereof.

In still another aspect, provided herein is a pharmaceutical composition comprising a compound of formula (III):

or a pharmaceutically acceptable salt therof.

In one embodiment, the pharmaceutical composition further comprises ceftolozane sulfate. In another embodiment, the pharmaceutical composition further comprises tazobactam.

In another embodiment, the compound of formula (III) is obtained by a process comprising the step of reacting ceftolozane and formylacetic acid to obtain the compound of formula (III). In another embodiment, the compound of formula (III) is obtained by a process comprising the step of reacting ceftolozane and tazobactam acid to obtain the compound of formula (III).

In another embodiment, the compound of formula (III) is obtained by a process comprising the steps of: forming an aqueous solution comprising ceftolozane and tazobactam acid; and lyophilizing the aqueous solution to obtain a lyophilized composition comprising the compound of formula (III). In another embodiment, the aqueous solution comprises ceftolozane sulfate and tazobactam acid in a 2:1 weight ratio between the amount of ceftolozane active and the amount of tazobactam acid. In another embodiment, the aqueous solution comprises sodium chloride, ceftolozane sulfate, tazobactam acid and L-arginine.

In another embodiment, the aqueous solution has a pH of about 5.0 to 7.0, e.g., 6.0 to 7.0. In another embodiment, the pharmaceutical composition is formulated for parenteral administration. In another embodiment, the compound of formula (III) is obtained by a process further comprising the step of performing high performance liquid chromatography (HPLC) on the lyophilized composition to isolate the compound of formula (III).

In another embodiment, the pharmaceutical composition comprises about 0.13-0.97% of the compound of formula (III). In another embodiment, the pharmaceutical composition comprises up to about 0.15% of the compound of formula (III). In another aspect, provided herein is a pharmaceutical composition comprising a compound of formula (III),

the compound of formula (III) obtained by a process comprising the steps of: forming an aqueous solution comprising tazobactam acid and ceftolozane sulfate in an amount providing 1,000 mg of ceftolozane active per 500 mg of tazobactam acid in the aqueous solution; lyophilizing the aqueous solution of step (a) to obtain a lyophilized composition comprising a compound of formula (III); and formulating the lyophilized composition as a pharmaceutical composition for parenteral delivery.

In one embodiment, the pH of the aqueous solution is 5.0 to 7.0, e.g., 6.0 to 7.0. In another embodiment, the pharmaceutical composition is formulated for parenteral administration.

In another aspect, provided herein is a pharmaceutical composition formulated for parenteral administration for the treatment of complicated intra-abdominal infections or complicated urinary tract infections, the pharmaceutical composition comprising a compound of formula (III) in a lyophilized composition obtained by lyophilizing an aqueous solution comprising tazobactam and an amount of ceftolozane sulfate containing 1,000 mg of ceftolozane active per 500 mg of tazobactam acid.

B. Co-Lyophilization

In one aspect, provided herein is a method of preparing a composition comprising ceftolozane and sodium chloride, comprising combining sodium chloride with ceftolozane, wherein 125-1000 mg sodium chloride per 1000 mg of ceftolozane is combined, followed by lyophilization of the sodium chloride ceftolozane mixture. The process is referred to herein as “co-lyophilization”. In another aspect, provided herein is a method of preparing a composition comprising sodium chloride, tazobactam, and ceftolozane, comprising combining sodium chloride, tazobactam, and ceftolozane, wherein 125-1000 mg sodium chloride per 1000 mg of ceftolozane is combined, followed by lyophilization of the mixture of sodium chloride, tazobactam, and ceftolozane.

Also provided herein is a method of preparing a pharmaceutical composition comprising sodium chloride, ceftolozane, and tazobactam, comprising combining sodium chloride, tazobactam, and ceftolozane, followed by spray-drying the mixture of sodium chloride, ceftolozane, and tazobactam.

FIG. 2 is a flowchart showing the steps for preparing a CXA-201 composition comprising ceftolozane (referred to as CXA-101) and tazobactam using a co-lyophilization process, as described herein.

In another aspect, provided herein is a pharmaceutical composition comprising stabilized ceftolozane sulfate obtained by a process comprising lyophilizing an aqueous solution comprising 125 mg to 500 mg sodium chloride with an amount of ceftolozane sulfate providing 1,000 mg of ceftolozane active, to obtain the lyophilized stabilized ceftolozane sulfate composition.

In one embodiment, the stabilized ceftolozane is obtained by lyophilizing the sodium chloride and ceftolozane sulfate with L-arginine. In another embodiment, the stabilized ceftolozane is obtained by lyophilizing an aqueous solution having a pH of about 5.0 to 7.0, e.g., 6.0 to 7.0.

In another embodiment, the stabilized ceftolozane is obtained by lyophilizing the sodium chloride and ceftolozane sulfate with L-arginine and citric acid. In another embodiment, the pharmaceutical composition is formulated for parenteral administration. In another embodiment, the composition is a unit dosage form in a container comprising 125 mg to 500 mg sodium chloride, 1,000 mg of ceftolozane in the form of ceftolozane sulfate, and L-arginine. In another embodiment, the pharmaceutical composition is formulated for parenteral administration. In another embodiment, the pH of the aqueous solution is 6.0 to 7.0.

In another aspect, provided herein is a container comprising a pharmaceutical composition of stabilized ceftolozane sulfate, obtained by a process comprising the step of: lyophilizing an aqueous solution comprising 125 mg to 500 mg sodium chloride with an amount of ceftolozane sulfate providing 1,000 mg of ceftolozane active, to obtain the lyophilized stabilized ceftolozane sulfate composition; filling the lyophilized stabilized ceftolozane composition into a container.

IV. Manufacturing For the Prevention of Cross-Contamination

Recent FDA manufacturing guidance (published in April 2013) states that manufacturing facilities dedicated to manufacturing a sensitizing non-penicillin beta-lactam compound should be “completely and comprehensively separated” from areas in the facility in which any class of sensitizing beta-lactam is manufactured. See U.S. Department of Health and Human Services Food and Drug Administration, Center for Drug Evaluation and Research, Non-Penicillin Beta-Lactam Drugs: A CGMP Framework for Preventing Cross-Contamination (April 2013) (“FDA Guidance”). The FDA also considers separation of production facilities for penicillins to be good manufacturing practice. The FDA Guidance can be understood to require the use of a dedicated facility to manufacture antibiotic compounds comprising a non-penicillin beta-lactam compound (e.g., a cephalosporin) and a BLI compound with a beta-lactam ring (e.g., tazobactam). Accordingly, a facility that manufactures a product containing both cephalosporin and a beta-lactam containing BLI such as tazobactam for sale in the United States cannot be subsequently used to manufacture any other products containing beta-lactam ring, other than additional combinations of other cephalosporins with the same BLI compound (e.g., other non-penicillin beta-lactam compounds including other cephalosporin antibitoics cannot be subsequently manufactured in the facility).

Beta-lactam antibiotics, including penicillin and the non-penicillin classes, share a basic chemical structure that includes a three-carbon, one-nitrogen cyclic amine structure known as the beta-lactam ring. The side chain associated with the beta-lactam ring is a variable group attached to the core structure by a peptide bond; the side chain variability contributes to antibacterial activity. As of the date of this publication, FDA has approved over 34 beta-lactam compounds as active ingredients in drugs for human use. (see, e.g., FDA's Approved Drug Products with Therapeutic Equivalence Evaluations, generally known as the Orange Book) Beta-lactam antibiotics include the following five classes: penicillins (e.g., ampicillin, oxacillin); cephalosporins (e.g., cephalexin, cefaclor); penems (e.g., imipenem, meropenem); carbacephems (e.g., loracarbef); and monobactams (e.g., aztreonam). (Yao, J D C, and R C Moellering, Jr., Antibacterial agents, in Manual of Clinical Microbiology, 9th edition, edited by PR Murray et al., Washington D.C., ASM Press, 2007.)

Under the FDA Guidance, a manufacturing facility handling a product for sale in the United States containing both a cephalosporin (e.g, ceftolozane) and a penicillin nucleus (e.g., tazobactam) cannot be subsequently used in the manufacture of any other class of beta-lactam products, including all other penicillins, cephalosporins, penems, carbacephems and monobactams or in the manufacture of other finished pharmaceuticals or active pharmaceutical ingredients. The FDA Guidance states that (non-penicillin) cephalosporin beta-lactam compounds (e.g., such as ceftolozane) for sale in the United States must be “completely and comprehensively separated from” manufacturing areas that handle any other class of beta-lactam compound (e.g., compounds in the penicillin class).

A product containing ceftolozane and tazobactam includes both a non-penicillin beta-lactam cephalosporin (ceftolozane) and a beta-lactamase inhibitor with a beta-lactam moiety (tazobactam). Under the FDA Guidance, these two compounds must be “completely and comprehensively separated.” Accordingly, there is a need for methods of manufacturing antibiotic compositions comprising ceftolozane and tazobactam for sale in the United States in compliance with the FDA Guidance, as well as antibiotic compositions manufactured in accordance with the FDA Guidance without affecting the purity, stability, and safety of the resulting composition.

Provided herein are methods of manufacturing or preparing pharmaceutical compositions containing two or more beta-lactam compounds in accordance with FDA Guidance, as well as pharmaceutical compositions manufactured in compliance with FDA Guidance. Specifically, certain manufacturing methods are provided herein that conform to standards recommended by FDA Guidance for the avoidance of cross-contamination of non-penicillin beta-lactam drugs.

In one aspect, provided herein is an antibacterial pharmaceutical composition formulated for parenteral administration for the treatment of infections, the pharmaceutical composition comprising a therapeutically effective amount of ceftolozane sulfate and tazobactam in a ratio of 1,000 mg ceftolozane active per 500 mg of tazobactam active, the pharmaceutical composition obtained by a process comprising the steps of:

-   -   a. lyophilizing a first aqueous solution in the absence of         tazobactam, the first aqueous solution comprising ceftolozane         sulfate, to obtain a first lyophilized ceftolozane composition;     -   b. blending the lyophilized ceftolozane composition with a         tazobactam composition comprising tazobactam prepared and         provided in the absence of ceftolozane;

wherein the process is completed in the absence of other non-cephalosporin beta-lactam compounds.

In another aspect, provided herein is a unit dosage form of a pharmaceutical composition formulated for parenteral administration for the treatment of complicated intra-abdominal infections or complicated urinary tract infections, the pharmaceutical composition comprising ceftolozane sulfate and tazobactam in a ratio of 1,000 mg ceftolozane active per 500 mg of tazobactam active, the pharmaceutical composition obtained by a process comprising the steps of

-   -   a. lyophilizing a first aqueous solution in the absence of         tazobactam, the first aqueous solution comprising ceftolozane         sulfate, 125 mg to 500 mg of sodium chloride per 1,000 mg of         ceftolozane active, to obtain a first lyophilized ceftolozane         composition,     -   b. lyophilizing a second solution comprising tazobactam in the         absence of ceftolozane to form a second lyophilized tazobactam         composition; and     -   c. blending the first lyophilized ceftolozane composition and         the second lyophilized tazobactam composition to obtain the         antibacterial composition;

wherein the process is completed in the absence of other non-cephalosporin beta-lactam compounds.

V. Methods of Treatment

Pharmaceutical compositions comprising ceftolozane/tazobactam are being developed as an intravenous (IV) formulation for the treatment of complicated urinary tract infections (cUTIs) and complicated intra-abdominal infections (cIAIs).

Ceftolozane/tazobactam is an antibacterial composition including ceftolozane, a cephalosporin with potent antipseudomonal cephalosporinactivity, in combination with tazobactam, a beta (β)-lactamase inhibitor (BLI). Like other members of the cephalosporin class, ceftolozane is believed to exert its bactericidal activity by inhibiting essential penicillin-binding proteins (PBPs), resulting in inhibition of cell wall synthesis and subsequent cell death. Ceftolozane has activity against Pseudomonas aeruginosa including strains that are resistant to carbapenems, cephalosporins, fluoroquinolones, and aminoglycosides, and other common Gram-negative pathogens, including most extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae. Tazobactam inhibits chromosomal- and plasmid-mediated bacterial class A and C β lactamases. Tazobactam is believed to protect ceftolozane from hydrolysis by covalently binding these enzymes, and broadens coverage to include most ESBL-producing Escherichia coli, Klebsiella pneumoniae, and other Enterobacteriaceae, including some Enterobacteriaceae overexpressing AmpC. Tazobactam inhibits or decreases the activity of beta-lactamases (e.g., bacterial beta-lactamases), and can be combined with beta-lactam compounds (e.g., antibiotics), thereby broadening the spectrum of the beta-lactam compound and increasing the beta-lactam compound's efficacy against organisms that produce beta-lactamase. A compound or a composition possesses efficacy against an organism if it kills or weakens the organism, or inhibits or prevents reproduction the organism.

The CXA-201 product (ceftolozane/tazobactam for injection) is undergoing regulatory review for the treatment of complicated intra-abdominal infections (cIAI) caused by susceptible isolates of the Gram-negative and Gram-positive microorganisms such as: Citerobacter freundii, Escherichia coli, Enterobacter cloacae, Klebsiella pneumonia, Klebsiella oxytoca, Proteus mirabilis, and Pseudomonas aeruginosa. In patients who are at risk of mixed aerobic-anaerobic infection, concurrent therapy with an anti-anaerobic agent can also be used.

The CXA-201 product (ceftolozane/tazobactam for injection) is undergoing regulatory review for the treatment of complicated urinary tract infections (cUTI), including pyelonephritis caused by susceptible isolates of the following Gram-negative microorganisms: Enterobacter spp, Escherichia coli, Klebsiella pneumonia, Proteus mirabilis and Pseudomonas aeruginosa. In one embodiment, a pharmaceutical composition comprising a CXA-201 product (e.g, the unit dosage container of Table 29 below) is reconstituted in a pharmaceutically acceptable carrier (e.g., a total volume of about 90-150 mL, preferably about 110 mL, of 0.9% aqueous sodium chloride for injection or in initial volume of 10-20 mL of water for injection or 0.9% aqueous sodium chloride for injection, followed by dilution of this solution into a 100 mL volume of 0.9% aqueous sodium chloride for injection). The resulting pharmaceutical composition can be infused into a patient in need thereof for treatment of a complicated intra-abdominal infection (e.g, using 1 hour infusion times) three times per day (e.g., once every 8 hours) for a recommended duration of treatment (e.g. 4-10 days).

The preferred dosage of (ceftolozane/tazobactam for injection) for cUTI and cIAI is 1.5 g administered every 8 hours by intravenous (IV) infusion over 1 hour in patients ≧18 years of age. The duration of therapy should be guided by the severity and site of infection and the patient's clinical and bacteriological progress. In one embodiment, a pharmaceutical composition comprising a CXA-201 product (e.g, the unit dosage container of Table 29 below) is reconstituted in a pharmaceutically acceptable carrier (e.g., a total volume of about 90-150 mL, preferably about 110 mL, of 0.9% aqueous sodium chloride for injection or in initial volume of 10-20 mL of water for injection or 0.9% aqueous sodium chloride for injection, followed by dilution of this solution into a 100 mL volume of 0.9% aqueous sodium chloride for injection). The resulting pharmaceutical composition can be infused into a patient in need thereof for treatment of a Complicated Urinary Tract Infections (cUTI), Including Pyelonephritis (e.g, using 1 hour infusion times) three times per day (e.g., once every 8 hours) for a recommended duration of treatment (e.g. 7 days).

Ceftolozane/tazobactam displays potent antibacterial activity against common Gram-negative organisms, including Enterobacteriaceae and Pseudomonas aeruginosa; select Gram-positive organisms, including streptococci; the majority of pathogenic enteric bacilli and select Gram-positive anaerobic species, thus making ceftolozane/tazobactam a potentially practical choice for pathogens involved in gastro-intestinal, urinary and community acquired as well as nosocomial respiratory infections. In general, the Gram-positive and Gram-negative spectrum of ceftolozane is similar to ceftazidime, but its antipseudomonal activity is the most potent among all currently available β-lactams, including the cephalosporins and carbapenems. Most importantly, ceftolozane has been shown to be active against strains of P. aeruginosa that are resistant to carbapenems, cephalosporins, fluoroquinolones, and aminoglycosides, including the majority of multi-drug resistant isolates. Indeed, the minimum inhibitory concentration (MIC) required to inhibit the growth of 90% of organisms (MIC90) for P. aeruginosa (MIC90≦2 μg/mL) is the lowest among all systemically administered antipseudomonal antibiotics.

In vitro studies have demonstrated that ceftolozane/tazobactam has a broad spectrum of activity against Gram-negative bacteria. The in vitro activity of ceftolozane and ceftolozane/tazobactam was evaluated against a broad range of Gram-positive and Gram-negative bacteria. It was observed that tazobactam potentiated the activity of ceftolozane against Acinetobacter spp. and common species of Enterobacteriaceae, including Citrobacter spp., Enterobacter cloacae, E. coli, K. pneumoniae, Proteus mirabilis, and Serratia marcescens. These surveillance data demonstrate that 88% to 100% of these Enterobacteriaceae species are inhibited at <8 μg/mL.

In one aspect, provided herein is a method for the treatment of bacterial infections in a mammal, comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition prepared according to the methods described herein. In another aspect, provided herein is a method for the treatment of bacterial infections in a mammal, comprising administering to said mammal a therapeutically effective amount of tazobactam and ceftolozane. In certain embodiments of the above methods, the bacterial infection is caused by an extended-spectrum beta-lactamase-producing organism. In certain embodiments, the bacterial infection is caused by an antibiotic-resistant organism. In yet another aspect, the invention is a method for the treatment of bacterial infections in a mammal, comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising both tazobactam and ceftolozane. In certain embodiments of the above methods, the bacterial infection is caused by an extended-spectrum beta-lactamase-producing organism. In certain embodiments, the bacterial infection is caused by an antibiotic-resistant organism.

In another aspect, provided herein is a method for the treatment of bacterial infections in a mammal, comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising tazobactam, ceftolozane, and less than 0.1% by weight of the compound RRT 1.22. In another embodiment of the treatment method, the pharmaceutical composition comprises tazobactam, ceftolozane, and less than 0.05% by weight of the compound RRT 1.22.

In certain embodiments of the treatment methods, the pharmaceutical composition further comprises 125 to 1000 mg sodium chloride per 1000 mg of ceftolozane, e.g., 125 to 500 mg sodium chloride per 1000 mg of ceftolozane, 200-500 mg sodium chloride per 1000 mg of ceftolozane, 300-500 mg sodium chloride per 1000 mg of ceftolozane, 400-500 mg sodium chloride per 1000 mg of ceftolozane, 450-500 mg sodium chloride per 1000 mg of ceftolozane, 460-500 mg sodium chloride per 1000 mg of ceftolozane, or about 476 mg sodium chloride per 1000 mg of ceftolozane. In one specific embodiment of the treatment methods, the pharmaceutical composition further comprises about 487 mg sodium chloride per 1000 mg of ceftolozane.

In other embodiments of the treatment methods, the pharmaceutical composition comprises 250-750 mg tazobactam per 1000 mg of ceftolozane, e.g., 250-700 mg tazobactam per 1000 mg of ceftolozane, 300-700 mg tazobactam per 1000 mg of ceftolozane, 300-650 mg tazobactam per 1000 mg of ceftolozane, 350-650 mg tazobactam per 1000 mg of ceftolozane, 350-600 mg tazobactam per 1000 mg of ceftolozane, 400-600 mg tazobactam per 1000 mg of ceftolozane, 400-550 mg tazobactam per 1000 mg of ceftolozane, 450-550 mg tazobactam per 1000 mg of ceftolozane, or about 500 mg tazobactam per 1000 mg of ceftolozane.

Non-limiting examples of the bacterial infections that can be treated by the methods of the invention include infections caused by: aerobic and facultative gram-positive microorganisms (e.g., Staphylococcus aureus, Enterococcus faecalis, Staphylococcus epidermidis, Streptococcus agalactiae, Streptococcus pneumonia, Streptococcus pyogenes, Viridans group streptococci), aerobic and facultative gram-negative microorganisms (e.g., Acinetobacter baumanii, Escherichia coli, Haemophilus influenza, Klebsiella pneumonia, Pseudomonas aeruginosa, Citrobacter koseri, Moraxella catarrhalis, Morganella morganii, Neisseria gonorrhoeae, Proteus mirabilis, Proteus vulgaris, Serratia marcescens, Providencia stuartii, Providencia rettgeri, Salmonella enterica), gram-positive anaerobes (Clostridium perfringens), and gram-negative anaerobes (e.g., Bacteroides fragilis group (e.g., B. fragilis, B. ovatus, B. thetaiotaomicron, and B. vulgates), Bacteroides distasonis, Prevotella melaninogenica).

In certain embodiments of the methods described herein, the bacterial infections resulting from beta-lactamase-producing organisms are treated or controlled. Non-limiting examples of beta-lactamase-producing organisms include:

(1) ESBL (extended-spectrum beta-lactamase)-producing organisms selected from the group consisting of Enterobacteriaceae spp.: Escherichia coli, Klebsiella spp. (including K. pneumoniae and K. oxytoca), Proteus mirabilis, Proteus vulgaris, Enterobacter spp., Serratia spp., Citrobacter spp., Pseudomonas spp., Acinetobacter spp.) and Bacteroides spp.;

(2) CSBL (conventional-spectrum beta-lactamase)-producing organisms, known to those of skill in the art; and

(3) Inducible-AmpC-type beta-lactamases, such as Citrobacter spp., Serratia spp., Morganella morganii, Proteus vulgaris, and Enterobacter cloacae.

In certain embodiments of the methods described herein, the bacterial infection is associated with one or more of the following conditions:

Appendicitis (complicated by rupture or abscess) and peritonitis caused by piperacillin-resistant beta-lactamase producing strains of Escherichia coli or the following members of the Bacteroides fragilis group: B. fragilis, B. ovatus, B. thetaiotaomicron, or B. vulgates;

Uncomplicated and complicated skin and skin structure infections, including cellulitis, cutaneous abscesses, and ischemic/diabetic foot infections caused by piperacillin-resistant, beta-lactamase producing strains of Staphylococcus aureus;

Postpartum endometritis or pelvic inflammatory disease caused by piperacillin-resistant, beta-lactamase producing strains of Escherichia coli;

Community-acquired pneumonia (moderate severity only) caused by piperacillin-resistant, beta-lactamase producing strains of Haemophilus influenza;

Nosocomial pneumonia (moderate to severe) caused by piperacillin-resistant, beta-lactamase producing strains of Staphylococcus aureus and by Acinetobacter baumanii, Haemophilus influenzae, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Nosocomial pneumonia is also known as hospital acquired/ventilator-associated bacterial pneumonia (HABP/VABP);

Complicated intra-abdominal infections (cIAI);

Complicated urinary tract infections (cUTIs);

Acute Pyelonephritis; and

Systemic Inflammatory Response Syndrome (SIRS).

Also provided herein is the use of tazobactam, and hydrates and solvates thereof, in combination with ceftolozane, for the preparation of a medicament for the treatment of bacterial infections. The bacterial infections can result from either gram-negative or gram-positive organisms.

The compositions provided herein can be used in the treatment of infections caused by Pseudomonas aeruginosa, Serratia marcescens, Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae, or Streptococcus pneumonia.

In one embodiment of the treatment methods, the bacterial infections are Gram-negative bacterial infections. In one embodiment, the gram-negative infections are complicated Urinary Tract Infections (cUTI) and complicated intra-abdominal infections (cIAI). In another embodiment, the gram-negative bacterial infections are caused by Pseudomonas aeruginosa, E. coli, and/or Klebsiella pneumonia.

In a further embodiment, provided herein is a method for the treatment of gram-negative bacterial infections in a mammal, comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising ceftolozane, about 500 mg tazobactam free acid per 1000 mg of ceftolozane, about 476 mg sodium chloride per 1000 mg of ceftolozane, about 587 mg L-arginine per 1000 mg of ceftolozane, and about 21 mg anhydrous citric acid per 1000 mg of ceftolozane. In one embodiment, the gram-negative bacterial infections are selected from the group consisting of complicated Urinary Tract Infections (cUTI) and complicated intra-abdominal infections (cIAI). In another embodiment, the gram negative bacterial infection is nosocomial pneumonia.

In another specific embodiment, provided herein is a method for the treatment of gram-negative bacterial infections in a mammal, comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising ceftolozane, about 500 mg tazobactam free acid equivalents per 1000 mg of ceftolozane, about 487 mg sodium chloride per 1000 mg of ceftolozane, about 600 mg L-arginine per 1000 mg of ceftolozane, and about 21 mg anhydrous citric acid per 1000 mg of ceftolozane. In one embodiment, the gram-negative bacterial infections are selected from the group consisting of complicated Urinary Tract Infections (cUTI) and complicated intra-abdominal infections (cIAI). In another embodiment, the gram negative bacterial infection is nosocomial pneumonia.

In one embodiment, provided herein is a method for the treatment of an infection in a mammal, wherein the infection is caused by Pseudomonas aeruginosa, Serratia marcescens, Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae, or Streptococcus pneumoniae comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising ceftolozane, corresponding to approximately 1000 mg of the free base form of ceftolozane; tazobactam, corresponding to approximately 500 mg of the tazobactam acid form; and 400-500 mg sodium chloride. In one embodiment, the pharmaceutical composition further comprises 500-650 mg L-arginine and 15-30 mg anhydrous citric acid.

In another embodiment, provided herein is a method for the treatment of urinary tract infection, intra-abdominal infection, or nosocomial pneumonia in a mammal, comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising ceftolozane, corresponding to approximately 1000 mg of the free base form of ceftolozane; tazobactam, corresponding to approximately 500 mg of the tazobactam acid form; and 400-500 mg sodium chloride. In an embodiment, the pharmaceutical composition comprises 487 mg sodium chloride. In one embodiment, the pharmaceutical composition further comprises 500-650 mg L-arginine and 15-30 mg anhydrous citric acid.

In one embodiment, provided herein is a method for the treatment of an infection in a mammal, wherein the infection is caused by Pseudomonas aeruginosa, Serratia marcescens, Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae, or Streptococcus pneumoniae comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising approximately 1147 mg ceftolozane sulfate; approximately 537 mg tazobactam sodium; and 400-500 mg sodium chloride. In one embodiment, the pharmaceutical composition further comprises 500-650 mg L-arginine and 15-30 mg anhydrous citric acid.

In another embodiment, provided herein is a method for the treatment of urinary tract infection, intra-abdominal infection, or nosocomial pneumonia in a mammal, comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising approximately 1147 mg ceftolozane sulfate; approximately 537 mg tazobactam sodium; and 400-500 mg sodium chloride. In an embodiment, the pharmaceutical composition comprises 487 mg sodium chloride. In one embodiment, the pharmaceutical composition further comprises 500-650 mg L-arginine and 15-30 mg anhydrous citric acid.

In one embodiment, provided herein is a method for the treatment of an infection in a mammal, wherein the infection is caused by Pseudomonas aeruginosa, Serratia marcescens, Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae, or Streptococcus pneumoniae comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising approximately 1147 mg ceftolozane sulfate; approximately 537 mg tazobactam sodium; approximately 487 mg sodium chloride; and approximately 600 mg L-arginine. In one embodiment, the pharmaceutical composition further comprises 15-30 mg anhydrous citric acid.

As used herein, “treating”, “treat” or “treatment” describes the management and care of a patient for the purpose of combating a disease, condition, or disorder and includes the administration of a pharmaceutical composition of the present invention to alleviate the symptoms or complications of a disease, condition or disorder, or to eliminate the disease, condition or disorder. The term “treat” can also include treatment of a cell in vitro or an animal model.

By a “therapeutically effective amount” of a compound of the invention is meant a sufficient amount of the compound to treat the disorder (e.g., bacterial infection). The specific therapeutically effective amount that is required for the treatment of any particular patient or organism (e.g., a mammal) will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound or composition employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see, for example, Goodman and Gilman's, “The Pharmacological Basis of Therapeutics”, Tenth Edition, A. Gilman, J. Hardman and L. Limbird, eds., McGraw-Hill Press, 155-173, 2001, which is incorporated herein by reference in its entirety). The therapeutically effective amount for a given situation can be readily determined by routine experimentation and is within the skill and judgment of the ordinary clinician.

As used herein, “189 mg sodium from sodium chloride per 1000 mg of ceftolozane” refers to a ratio of sodium from the sodium chloride to ceftolozane active. For example, “189 mg sodium from sodium chloride per 1000 mg of ceftolozane” includes, for example, 94.5 mg sodium from sodium chloride per 500 mg of ceftolozane, as well as, for example, 47.25 mg sodium from sodium chloride per 250 mg ceftolozane. In addition, “1,000 mg of ceftolozane as ceftolozane sulfate” refers to an amount of ceftolozane sulfate effective to provide 1,000 mg of ceftolozane. “189 mg sodium from sodium chloride” refers to the amount of sodium chloride (e.g., 480 mg) effective to provide 189 mg of sodium. The amount of sodium from sodium chloride per gram of ceftolozane activity in a pharmaceutical composition containing ceftolozane sulfate, chloride and sodium chloride can be calculated using the relevant molecular weights of ceftolozane, ceftolozane sulfate, sodium chloride and sodium. For example, a composition comprising about 1,147 mg ceftolozane sulfate and 189 mg sodium from sodium chloride contains 480 mg sodium chloride per 1,000 mg ceftolozane active.

Unless otherwise indicated, as used herein, the term “Related Substances” with respect to HPLC detection refers to all the ceftolozane related process impurities and degradation products other than ceftolozane separated and detected by HPLC according to Example 1. Unless otherwise indicated, as used herein, the term “% Related Substances” refers to the % of the total HPLC peak area obtained by Example 1 attributed to all the ceftolozane related process impurities and degradation products other than ceftolozane.

EXAMPLES Example 1 HPLC Analysis of Compositions Comprising Ceftolozane

The purity of ceftolozane in the pharmaceutical compositions was measured using the analytical HPLC method described below.

The HPLC methodologies described herein were used to acquire the data provided in Examples 5 and 8.

Analytical HPLC Method A. Operative Conditions

Column Develosil ODS-UG-5; 5 μm, 250 × 4.6 mm (Nomura Chemical, Japan) Mobile phase Sodium perchlorate buffer solution (PH 2.5)/CH₃CN 90:10 (v/v) Flow rate 1.0 mL/min Wavelength 254 nm Injection volume 10 μl Oven Temperature 45° C. Run Time 85 minutes

Gradient Profile:

Time (min) A% B% 0 75 25 30 70 30 60 0 100 85 0 100 85.1 75 25 110 75 25

B. Mobile Phase Preparation.

Sodium perchlorate buffer solution was made by dissolving 14.05 g of sodium perchlorate monohydrate in 1000.0 mL of water followed by adjusting pH to 2.5 with diluted perchloric acid (1 in 20).

Mobile phase was then made by mixing sodium perchlorate buffer solution (pH 2.5) and acetonitrile in the ratio 90:10 (v/v).

Sodium acetate buffer solution pH 5.5 (diluent) was made by dissolving 1.36 g of sodium acetate trihydrate in 1000.0 mL of water followed by adjusting to pH 5.5 with diluted acetic acid (1 in 10).

C. Sample Preparation.

Sample solution: dissolve 20.0 mg, exactly weighed, of the Sample, in 20.0 mL of water (Prepare just before injection into HPLC system).

System suitability solution (1%): take 1.0 mL of the sample solution (the first sample if more are present) and transfer into a 100.0 mL volumetric flask, dilute with water to volume and mix.

D. HPLC Analysis Procedure

-   1. Inject blank (water) -   2. Inject system suitability solution and check for tailing factor     and theoretical plate number for the CXA-101 peak:     -   The tailing factor must not be greater than 1.5     -   Theoretical plates number must not be less than 10000 -   3. Inject sample solution -   4. Inject system suitability solution and check for tailing factor     and theoretical plate number for the CXA-101 peak.     -   The tailing factor must not be greater than 1.5     -   Theoretical plates number must not be less than 10000 -   5. Identify the peaks of related substances in the sample     chromatogram based on the reference chromatogram reported in FIG. 3     or, alternatively, on the basis of the following RRT values listed     in Table 1.

TABLE 1 Identities and RRTs of the Products Related to Ceftolozane Compound RRT Proposed Structure Source Peak 1 3-side chain ~0.14

Degradation product and process product Peak 2 ~0.16 Unidentified Process product Peak 3 ~0.4 Unidentified Process product Peak 4 ~0.6 Unidentified Process product Peak 5 7-Epimer type ~0.9

Degradation product and process product Peak 6 ~1.1 NA Process product Peak 7 Δ3 Isomer type ~1.30

Degradation product and process product Peak 8 ~1.37 Unidentified Process product Peak 9 Anti-Isomer type ~1.7

Process product and Degradation product Peaks 10, 11 ~2.3 Unidentified Process product

E. Calculations

I. Report for each related substance its amount as expressed by area percent.

$C_{i} = \frac{A_{i} \times 100}{A_{t} + {\sum A_{i}}}$

wherein:

C_(i)=Amount of related substance i in the sample, area %

A_(i)=Peak area of related substance i in the sample chromatogram

A_(t)=Area of CXA-101 peak in the sample chromatogram

A_(t)+ΣA_(i)=Total peaks area in the sample chromatogram

Consider as any unspecified compound, each peak in the chromatogram except CXA-101, peaks from 1 to 11 and every peak present in the blank chromatogram and report the largest.

II. Report the total composition content as expressed by the following formula:

$C_{T} = \frac{A_{i} \times 100}{A_{t} + {\sum A_{i}}}$

wherein:

C_(T)=total composition content in the sample, area %

A_(t)=area of CXA-101 peak in the sample chromatogram

ΣA_(i)=total peak areas of composition in the sample chromatogram

FIG. 3 is a reference HPLC chromatogram showing the peaks of ceftolozane (CXA-101) and related composition peaks.

Example 2 Screening of Stabilizing Agents

Nine stabilizing agents were screened, including sodium chloride, fructose, xylitol, sorbitol, dextran 40, lactose, glucose, maltose, and D-mannitol. The purity of the ceftolozane in a composition comprising 100 mg ceftolozane and 100 mg of one of the stabilizing agents after 3 days at 70° C. was compared to a composition comprising 100 mg ceftolozane but no stabilizing agent.

As shown in Table 2, the ceftolozane compositions comprising sodium chloride, dextran 40, lactose, or maltose were demonstrated to be more stable than the other ceftolozane compositions comprising the other stabilizing agents, or no stabilizing agent. Sodium chloride and maltose were selected for further investigation.

TABLE 2 Screening of Stabilizing Agents Stabilizing agent Sodium chloride Fructose Xylitol Sorbitol Dextran 40 Storage 70° C. 70° C. 70° C. 70° C. 70° C. Initial 3 days Initial 3 days Initial 3 days Initial 3 days Initial 3 days Appearance White Pale White Orange White Orange White Pale White Pale mass yellow mass paste mass paste mass yellow mass yellow mass paste mass Color and Pale Pale Pale Orange Pale Orange Pale Yellow Pale Yellow clarity yellow yellow yellow and yellow and yellow and yellow and and and and clear and clear and clear and clear clear clear clear clear clear clear pH 5.58 4.23 6.04 3.81 5.96 4.18 6.01 4.00 5.60 4.36 Residual rate 100.0 75.7 100.0 4.29 100.0 0.41 100.0 0.00 100.0 72.2 (%) Reconstitution 15 30 20 40 15 180<    15 160 170 160 time(s) Stabilizing agent No stabilizing agent Lactose Glucose Maltose D-Mannitol (Control) Storage 70° C. 70° C. 70° C. 70° C. 70° C. Initial 3 days Initial 3 days Initial 3 days Initial 3 days Initial 3 days Appearance Pale Pale White Pale White Pale White Pale Pale Pale yellow yellow mass yellow mass yellow mass yellow yellow yellow mass mass mass mass mass mass mass Color and Pale Pale Pale Pale Pale Pale Pale Yellow Pale Yellow clarity yellow yellow yellow yellow yellow yellow yellow and yellow and and and and and and and and clear and clear clear clear clear clear clear clear clear clear pH 5.86 4.70 6.23 4.32 6.08 5.06 6.13 3.97 5.10 4.02 Residual rate 100.0 80.5 100.0 37.3 100.0 80.9 100.0 1.38 100.0 51.2 (%) Reconstitution 15 15 15 15 15 15 30 50 15 15 time(s)

The stabilizing effect of other non-reducing sugars such as sucrose and trehalose, as well as polyvinylpyrrolidone (PVP), was also evaluated in a ceftolozane formulation.

Five samples were prepared, the components of which are shown in Table 2a below. Each sample contained 1000 mg of ceftolozane active, 40 mg citric acid monohydrate (equivalent of 36 mg citric acid anhydrous), and the same amount of L-arginine. Stabilizing reagents in four samples are 480 mg sodium chloride, 300 mg of trehalose, 300 mg of sucrose, and 300 mg of PVP, respectively. One sample was a control that contained no stabilizing reagent. The samples were in lyophilized form and stored at 60° C. for 7 days. The purities of the samples were monitored by HPLC on day 0, day 1, day 3 and day 7.

TABLE 2a Comparison between stabilizing reagents Excipient NaCl Trehalose Sucrose PVP None Ceftolozane amount 1000 1000 1000 1000 1000 Excipient amount 480 300 300 300 N/A Purity: t₀ 98.42 98.09 98.14 97.89 97.94 60° C./1 d 97.85 96.73 96.97 96.05 96.15 60° C./3 d 97.21 95.36 95.81 94.57 94.53 60° C./7 d 95.65 94.21 94.19 92.78 92.06 Purity Δ (0-7 d) −2.77% −3.88% −3.95% −5.11% −5.88%

As shown in Table 2a, the sample containing sodium chloride exhibited the best stability. The purity of ceftolozane in the sample containing sodium chloride had the slightest purity drop over 7 days. This experiment further supports the discovery that sodium chloride provides surprisingly better stabilizing effect than the other reagents.

Example 3 Stability Study of Ceftolozane Compositions Comprising Sodium Chloride, or Maltose, or No Stabilizing Agent

Three ceftolozane compositions were prepared, the components of which are shown in Table 3. These compositions were put in a stressed stability study at 70° C. for 3 days and 6 days. The purity of the ceftolozane in the compositions was analyzed using the HPLC method described in Example 1.

TABLE 3 Ceftolozane Compositions CEF/no stabilizer 9.5 g active Ceftolozane 5.7 g L-Arginine 200 mg Citric acid CEF/maltose 9.5 g active Ceftolozane 5.7 g L-Arginine 200 mg Citric acid 5 g Maltose H₂O CEF/sodium chloride 9.5 g active Ceftolozane 5.7 g L-Arginine 200 mg Citric acid 4.6 g Sodium Chloride

The results are shown in Table 4 where only the most significant composition peaks (P1, P7, and P12) are shown. It was found that the composition comprising maltose (CEF/maltose) contained a significantly large amount of the composition P12 peak, which was identified as having the following formula:

In addition, the presence of maltose produced a particularly aggregated powder after lyophilization, which has a potentially negative impact to manufacturing ceftolozane compositions.

In contrast, the ceftolozane composition comprising sodium chloride (CEF/sodium chloride) was much more stable than the ceftolozane composition comprising maltose or the ceftolozane composition comprising no stabilizing agent. Therefore, sodium chloride was, unexpectedly, a better stabilizing agent for ceftolozane compositions.

TABLE 4 Stability Study of Ceftolozane Compositions Comprising Sodium Chloride, or Maltose, or No Stabilizing Agent CEF/no stabilizer P1 P7 P12 Total Time (days) 0 0.49 0.69 0.00 1.98 3 3.06 1.29 0.00 8.48 6 4.11 1.49 0.00 10.83 CEF/maltose P1 P7 P12 Total Time (days) 0 0.41 0.65 0.15 1.91 3 2.85 1.02 3.44 10.08 6 3.45 1.12 4.01 11.65 CEF/sodium chloride P1 P7 P12 Total Time (days) 0 0.20 0.62 0.00 1.64 3 1.70 0.85 0.00 4.29 6 2.86 1.05 0.00 6.70

Example 4a Manufacturing Procedure of Mono Product for Injection 4a.1. Preparation of the Compound Solution of CXA-101 Lyophilized Product

1) Weigh 30 kg of water for injection into the compounding vessel;

2) Add 100 g of citric acid, anhydrous and 150 g of sodium bicarbonate into the compounding vessel and dissolve them with mixing;

3) Weigh 5,000 g potency of CXA-101 drug substance and suspend it with mixing. (Note any generation of carbon dioxide.)

4) Slowly add 1,100 g of sodium bicarbonate and dissolve CXA-101 with mixing. (Again, note any generation of carbon dioxide.)

5) Add 1,146 g of sodium chloride and 10,000 g of maltose, dissolve with mixing.

6) Purge dissolved carbon dioxide in the solution with nitrogen until the pH of the solution does not change.

7) Adjust the pH of the solution to 6.0±0.1 with 5%-sodium bicarbonate solution.

8) Adjust the total weight to 56,850 g (D₂₀=1.137) with water for injection.

9) Confirm the pH of the compounded solution within the range 6.0±0.1.

4a.2. Prefiltration and Sterile-Filtration

10) Filtrate the compounded solution with a sterile tilter-set which consists of a 0.2 um polyvinylidene fluoride membrane filter (Durapore®, Millipore) and a 0.1 urn polyvinylidene fluoride membrane filter (Durapore®, Millipore) connected in tandem. Confirm the integrity of each filter before and after the filtration. Take approximately 100 mL of the filtrate in order to check bioburden.

11) Filter the prefiltered compounded solution through a sterile filter-set which consists of a 0.2 um polyvinylidene fluoride membrane filter and a 0.1 urn polyvinylidene fluoride membrane filter connected in tandem, and introduce the final filtrate into an aseptic room. Confirm the integrity of each filter before and after the filtration.

4a.3. Processing of Container, Stopper and Flip-Off Cap

12) Wash a sufficient quantity of 28 mL containers with water for injection and sterilize the washed containers by a dry-heat sterilizer. Then transfer the sterilized containers into a Grade A area located in an aseptic room.

13) Wash a sufficient quantity of stoppers with, water for injection. Sterilize and dry the washed stoppers by steam sterilizer. Then transfer the sterilized stoppers into a Grade A area located in an aseptic room.

14) Sterilize a sufficient quantity of flip-off caps by steam sterilizer. Then transfer the sterilized flip-off caps into a Grade A or B area located in an aseptic room.

4a.4. Filling and Partially Stoppering

15) Adjust the fill weight of the filtered compounded solution to 11.37 g (corresponds to 10 mL of the compounded solution), then start filling operation. Check the filled weight in sufficient frequency and confirm it is in target range (11.37 g±1%, 11.26 to 11.43 g). When deviation from the control range (11.37 g±2%, 11.14 to 11.59 g) is occurred, re-adjust the filling weight.

16) Immediately after a container is filled, partially stopper the container with a sterilized stopper. Load the filled and partially stoppered containers onto the shelves of a lyophilizer aseptically.

4a.5. Lyophilization to Crimping, Visual Inspection, Labeling and Packaging

17) After all filled and partially stoppered containers are loaded into a lyophilizer, start the lyophilization program. Freeze the loaded containers at −40° C. and keep until all containers freeze. Forward the program to primary drying step (shelf temperature; −20° C., chamber pressure; 100 to 150 mTorr). Primary drying time should be determined by monitoring the product temperature. Forward the program to secondary drying step (shelf temperature; 30° C., chamber pressure; not more than 10 mTorr) after completion of the primary drying step. After all containers are dried completely, return the chamber pressure to atmospheric pressure with sterilized nitrogen. Then stopper containers completely.

Example 4 Manufacturing Procedure of Bulk (Tray) Lyophilized Ceftolozane

There are four main steps in the manufacture of a CXA-101 pharmaceutical composition: dissolution, sterile filtration, bulk lyophilization, and packaging into Sterbags®. These four main steps are composed of a total of 20 minor steps. The flowchart of the manufacturing process is described below.

I. Dissolution

1. A prescribed amount of WFI (e.g., 81 kg WFI) is charged into a dissolution reactor.

2. A prescribed amount of citric acid (e.g., 20.7 mg anhydrous citric acid per ceftolozane active) is added.

3. The solution is cooled to 5° C. to 10° C.

4. A prescribed amount of CXA-101 drug substance (e.g., referenced to 1000 mg ceftolozane active) is added to the solution.

5. A prescribed amount of L-arginine (e.g., 587 mg L-arginine per 1000 mg ceftolozane active) is slowly added to the solution.

6. A check for complete dissolution is performed. Solution pH is verified to be in the target range of 6.5 to 7.0.

7. A prescribed amount of sodium chloride (e.g., 476 mg sodium chloride per 1000 mg ceftolozane active) is added to the solution.

8. A check for complete dissolution is performed. Solution pH is verified to be in the target range of 6.0 to 7.0. If the pH is out of this range, adjust with either L-Arginine or citric acid.

9. WFI is added to bring the net weight to 13.1 g and the solution is mixed well.

10. Samples are withdrawn for testing of final pH.

II. Sterile Filtration

11. The solution is passed through a filter (pore size 0.45 μm) followed by two more filters (pore size 0.22 μm) onto a shelf on the Criofarma lyophilizer.

12. The line is washed with WFI.

13. The washing solution from Step 12 is passed through sterile filtration.

III. Bulk Lyophilization

14. The washing solution is loaded onto a separate shelf on the lyophilizer (and later discarded).

15. The solution is lyophilized until dry.

16. The product shelf is cooled to 20° C.±5° C.

IV. Packaging into Sterbags®

17. The lyophilized pharmaceutical composition is milled.

18. The milled powder is sieved.

19. The sieved powder is blended for 30 minutes.

20. The powder is then discharged into Sterbags®

Example 5 Stabilizing Effect of Sodium Chloride in CXA-101 Compositions

A. Improvement in the Purity of the Ceftolozane in CXA-101 Pharmaceutical Compositions with Varying Amounts of Sodium Chloride

A stability study was carried out at 30° C. and 60° C. and analyzed by HPLC. The sodium chloride content in the CXA-101 compositions is described in Table 5. The HPLC data are summarized in Tables 6-9. The data are also plotted in FIGS. 4-7 to show the trends of the purity, and the amounts of the composition peak 1, the composition with a RRT of 0.43 and the composition peak 3, and the composition peak 7 in the CXA-101 compositions with respect to NaCl.

TABLE 5 Sodium Chloride Content in the CXA-101 Compositions Samples NaCl content A1 481.0 mg NaCl per 1000 mg of ceftolozane A2 190.0 mg NaCl per 1000 mg of ceftolozane A3 125.0 mg NaCl per 1000 mg of ceftolozane A4  75.0 mg NaCl per 1000 mg of ceftolozane A5  50.0 mg NaCl per 1000 mg of ceftolozane

TABLE 6 Purity of Ceftolozane in CXA-101 Compositions with Varying Amounts of Sodium Chloride Day A1 A2 A3 A4 A5 t0/60° C. 0 96.6 98.0 97.9 97.8 97.7 t0/30° C. 0 98.1 97.8 97.8 97.7 1 day/60° C. 1 95.9 96.9 96.5 95.7 95.5 1 day/30° C. 1 98.2 97.7 97.7 97.6 3 days/60° C. 3 94.9 95.7 94.8 93.9 93.6 (Δ_(t0-t3)) (1.7) (2.3) (3.1) (3.9) (4.1) 3 day/30° C. 3 98.0 97.5 97.5 97.3 7 days/60° C. 7 93.6 94.0 94.2 92.3 91.9 7 day/30° C. 7 97.8 97.2 97.1 97.0 Total Δ/60° C. 3.07 4.06 3.7 5.48 5.83 Total Δ/30° C. 0.3 0.6 0.7 0.7

TABLE 7 HPLC Peak Area of Composition Peak 1 in CXA-101 Compositions with Varying Amounts of Sodium Chloride Day A1 A2 A3 A4 A5 t0/60° C. 0 0.95 0.31 0.3 0.36 0.39 t0/30° C. 0 0.47 0.36 0.36 0.39 1 day/60° C. 1 1.36 0.86 0.94 1.36 1.39 1 day/30° C. 1 0.48 0.40 0.42 0.48 3 days/60° C. 3 1.71 1.31 1.73 2.06 2.1 3 day/30° C. 3 0.53 0.50 0.52 0.58 7 days/60° C. 7 2.26 2.14 2.07 2.86 2.93 7 day/30° C. 7 0.62 0.63 0.66 0.72 INCREASE %/60° C. 1.31 1.83 1.77 2.5 2.54 INCREASE %/30° C. 0.15 0.27 0.30 0.33

TABLE 8 HPLC Peak Area of the Composition with a RRT of 0.43 and Composition Peak 3 in CXA-101 Compositions with Varying Amounts of Sodium Chloride Day A1 A2 A3 A4 A5 t0/60° C. 0 0.28 0.10 0.09 0.10 0.11 t0/30° C. 0 0.15 0.10 0.10 0.11 1 day/60° C. 1 0.37 0.13 0.16 0.35 0.36 1 day/30° C. 1 0.13 0.09 0.09 0.10 3 days/60° C. 3 0.68 0.21 0.31 0.71 0.71 3 day/30° C. 3 0.17 0.13 0.13 0.14 7 days/60° C. 7 1.04 0.36 0.30 0.81 0.81 7 day/30° C. 7 0.19 0.16 0.16 0.17 INCREASE %/60° C. 0.76 0.26 0.21 0.71 0.7 INCREASE %/30° C. 0.04 0.06 0.06 0.06

TABLE 9 The HPLC Peak Area of Composition Peak 7 in CXA-101 Compositions with Varying Amounts of Sodium Chloride Day A1 A2 A3 A4 A5 t0/60° C. 0 1.31 0.95 0.96 1.01 1.02 t0/30° C. 0 0.69 1.00 1.01 1.02 1 day/60° C. 1 1.37 1.10 1.10 1.23 1.29 1 day/30° C. 1 0.68 0.99 1.01 1.02 3 days/60° C. 3 1.43 1.19 1.27 1.41 1.46 3 day/30° C. 3 0.68 1.03 1.01 1.05 7 days/60° C. 7 1.49 1.31 1.35 1.55 1.57 7 day/30° C. 7 0.68 1.01 1.03 1.07 INCREASE %/60° C. 0.18 0.36 0.39 0.54 0.55 INCREASE %/30° C. NC 0.01 0.02 0.05

Conclusion:

The stability test demonstrates that high sodium chloride content enhances stability of CXA-101 Compositions.

The HPLC measurements on day 3 were used to analyze the stability of the CXA-101 compositions.

CXA-101 compositions comprising high amounts of sodium chloride (e.g., 125-1000 mg sodium chloride per 1000 mg of ceftolozane) were found to be more chemically stable than CXA-101 compositions comprising low amounts of sodium chloride (e.g., less than 125 mg sodium chloride per 1000 mg of ceftolozane). Table 6 shows that, by day 3 of heating at 60° C., sample A1, which has the highest salt concentration, is most stable, i.e., has the lowest Δ_(t0-t3) of all samples. By day 3, the sample with the lowest salt concentration, A5, has the highest Δ_(t0-t3) indicating the most degradation. Overall, A5 has degraded 141% more than A1. Further, Table 6 shows that, by day 3 of heating at 60° C., sample A3, which contains a lower salt concentration within the limits of the invention at 125 mg, is still significantly more stable than A4, a composition containing 75.0 mg of the salt. A3 has a Δ_(t0-t3) of 3.1, while A4 has a Δ_(t0-t3) of 3.9, meaning that A4 has degraded 26% more than A3.

B. Long-Term Stability Study of CXA-101 Pharmaceutical Compositions with Varying Amounts of Sodium Chloride

Another stability study was carried out at 5° C. and 25° C. The sodium chloride content in the CXA-101 compositions is described in Table 9a. The amounts of citric acid and L-arginine in each composition were the same. These samples were in lyophilized form and were placed on long-term (24-36 months), real time stability programs.

The composition peak 1 is considered “diagnostic” for formulation failure because it is the first peak to go out of trend or specification (1.5%). Thus, the stability of these CXA-101 compositions was also measured by the length of storage until formulation failure as indicated by the composition peak 1. The data in Table 9a were extrapolated from data collected after 4 months. Clearly, based on the amount of the composition peak 1 in the compositions, the composition with about 480 mg sodium chloride per 1 gram ceftolozane active was significantly more stable than the compositions containing 125 mg or 62.5 mg sodium chloride per 1 gram of active ceftolozane (i.e., stability of ceftolozane compositions: 480>>125 mg>62.5 mg).

TABLE 9a The Peak 1 Failure Points of CXA-101 Compositions with Varying Amounts of Sodium Chloride Ceftolozane Peak 1 failure Peak 1 failure active, 1 g + point at 5° C. point at 25° C.  480 mg NaCl 245 months 15 months  125 mg NaCl  70 months  5 months 62.5 mg NaCl  25 months  3 months* *Results at 3 months = 1.34%, 4 months = 1.15%

Example 6 Manufacturing Process of a CXA-201 Composition Comprising Tazobactam and CXA-101/Ceftolozane by Co-Lyophilization

The manufacturing process of a CXA-201 composition comprising tazobactam and ceftolozane by co-lyophilization is shown in FIG. 2. Non-sterile bulk tazobactam and bulk ceftolozane were mixed, followed by dissolution and sterile filtration. The filtrate was then tray-lyophilized to obtain the CXA-201 composition. The CXA-201 composition can be container-filled as a final drug product. The components of a CXA-201 composition prepared by co-lyophilization are shown in Table 10.

TABLE 10 Components of a CXA-201 Composition Prepared by Co-lyophilization Component Function Amount (mg/container) Ceftolozane Active pharmaceutical ingredient 1000 (potency) L-arginine Alkalization reagent  587 Citric acid (anhydrous) Buffer  21 Sodium chloride Stabilizer  476 Tazobactam (free acid) Active pharmaceutical ingredient  500 Sodium bicarbonate Alkalization reagent Quantity sufficient¹ for pH 4.8 to 7.0 Water Dissolution solvent Not more than 4% by weight² Nitrogen Inert gas Sufficient quantity ¹Sodium content is approximately 78 mg/g of tazobactam in drug product after lyophilization. ²Water is removed during the lyophilization process and is controlled at no more than 4% by weight.

Example 7 Assessment of Co-Lyophilized Combo Drug Product (i.e., a CXA-201 Composition) A. Preparation of the Co-Lyophilized Combo Drug Product (i.e. the CXA-201 Composition)

The components of the co-lyophilized CXA-201 composition are shown in Table 11. This composition was prepared, as described above in Example 6.

TABLE 11 Components of the CXA-201 Composition Prepared by Co-Lyophilization CXA-201 16.3 g active ceftolozane Comp. 8.1 g active Tazobactam free ac. 15.5 g L-Arginine 350 mg Citric acid 7.9 g NaCl 6.1 pH compounded solution

B. Stressed Stability Test

Stability studies of this CXA-201 composition prepared by co-lyophilization were carried out at 25° C. and 40° C. The composition was analyzed using HPLC. The following Tables 12 and 13 are summaries of the HPLC measurements at time zero, after one month (T1), and after three months (T2).

TABLE 12 Stability Data of Co-Lyophilized CXA-201 Composition at 25° C./RH = 60% Test items Spec. D.P. T0 T1 25° C. T2 25° C. Related Substances Peak1 ≦1.50%   0.31%   0.54%   0.71% Peak2 ≦0.40%   0.07%   0.07%   0.09% Peak3 ≦0.30% <0.03% <0.03% <0.03% Peak4 ≦0.80%   0.08%   0.08%   0.09% Peak5 ≦1.00%   0.27%   0.26%   0.29% Peak6 ≦0.15% <0.03% <0.03% <0.03% Peak7 ≦2.00%   0.64%   0.65%   0.66% Peak8 ≦0.15% <0.03% <0.03% <0.03% Peak9 ≦0.60%   0.05%   0.11%   0.10% Peak10,11 ≦0.15% each   0.04%   0.04%   0.04% Peak12 ≦2.00% <0.03% <0.03% <0.03% Others (RRT 0.43) ≦0.15% <0.03% <0.03%   0.04% Others (RRT 1.22) ≦0.15%   0.13%   0.30%   0.38% Others (RRT 2.18) ≦0.15%   0.03% <0.03%   0.05% Others (RRT 2.77) ≦0.15% <0.03%   0.03%   0.03% Sing. Unk. ≦0.15%   0.05%   0.07%   0.05% Total ≦5.00%   1.67%   2.19%   2.77% pH report value   5.5      4.83

TABLE 13 Stability Data of Co-Lyophilized CXA-201 Composition at 40° C./RH = 75% Test items Spec. D.P. T0 T1 40° C. T2 40° C. Related Substances Peak1 ≦1.50%   0.31%   1.77%   2.22% Peak2 ≦0.40%   0.07%   0.10%   0.16% Peak3 ≦0.30% <0.03% <0.03%   0.06% Peak4 ≦0.80%   0.08%   0.09%   0.09% Peak5 ≦1.00%   0.27%   0.27%   0.30% Peak6 ≦0.15% <0.03% <0.03% <0.03% Peak7 ≦2.00%   0.64%   0.69%   0.78% Peak8 ≦0.15% <0.03% <0.03%   0.10% Peak9 ≦0.60%   0.05%   0.09%   0.09% Peak10,11 ≦0.15% each   0.04%   0.04%   0.05% Peak12 ≦2.00% <0.03% <0.03% <0.03% Others (RRT 0.43) ≦0.15% <0.03%   0.09%   0.15% Others (RRT 1.22) ≦0.15%   0.13%   0.74%   0.97% Others (RRT 2.18) ≦0.15%   0.03% <0.03%   0.08% Others (RRT 2.77) ≦0.15% <0.03% <0.03%   0.04% Sing. Unk. ≦0.15%   0.05%   0.11%   0.25% Total ≦5.00%   1.67%   4.49%   6.32% pH report value   5.5      4.09

C. Conclusion:

A new compound having RRT=1.22 was observed in the co-lyophilized CXA-201 compositions. While not wishing to be bound by theory, the compound RRT 1.22 was identified as a compound formed by a reaction between ceftolozane and formylacetic acid, which was a by-product of tazobactam as illustrated in Marunaka et al. (Chem. Pharm. Bull. 1988, Vol. 36 (11), pp. 4478-4487). The stability data at 25° C. and at 40° C. have confirmed the continued formation of the compound RRT 1.22 over the course of time.

Example 7a Identifying the Compound of Formula (III)

The Co-Lyophilized Combo Drug Product was prepared as described above in Example 6. The formulation composition of the Co-Lyophilized Combo drug product is shown in Table 11 (Example 7). This sample maintained at 25° C./RH=60% and 40° C./RH=75% after one month (T1) and three months (T2). Samples were analyzed using a HPLC method as described in Example 1. The data for analysis of the samples by HPLC is shown in Example 10 in Table 23 (Stability data of Co-Lyo Combo Drug Product at 25° C.) and Table 24 (Stability data Co-Lyo Combo Drug Product at 40° C.). The presence of the compound of Formula (III) was identified has having a retention time of about 1.22 as measured by HPLC (see Example 2). RRT=1.22 was observed in co-lyophilized drug product. The compound of formula (III) is believed to be formed by a reaction between ceftolozane and formylacetic acid, which was a degradation product of tazobactam. The amount of the compound of formula (III) in a composition comprising ceftolozane and tazobactam can be increased over time at 25° C. and at 40° C.

The material obtained from the RRT 1.22 peak was analyzed by LC/MS, providing the spectra shown in FIG. 14. FIG. 15 (below) is the corresponding structures for the peaks shown in FIG. 14.

A test sample prepared from ceftolozane and tazobactam acid co-compounding solution containing RRT 1.22 impurity was used on the LC/MS experiment. Liquid chromatography separation was performed on a Zorbax SB C8, 3.5 μm, 3.0 mm×150 mm column, using gradient elution with 20 mM ammonium formate containing 0.1% Heptofluorobutyric acid pH 3.2 as mobile phase A and 0.1% Heptofluorobutyric acid in acetonitrile as mobile phase B. The gradient starts from 3% (initial) to 15% mobile phase B in 20 minutes (with RRT 1.22 eluting at about 10.7 minutes). Mass detection was performed using electrospray ionization technique under positive mode. The column effluent was also monitored at 254 nm using a photodiode-array detector. MS/MS fragmentation was performed on m/z 737.3 positive ion using nitrogen as collision gas, with collision energy set at 35V.

Example 8 Stabilizing Effect of Sodium Chloride in CXA-201 Compositions A. Reduction of the Composition at RT=63 Minutes in CXA-201 Compositions

A stability study was carried out at 25° C. and analyzed by HPLC. CXA-201 compositions comprise ceftolozane and tazobactam, further comprising high, mid, or low amounts of sodium chloride (480, 125, or 62.5 mg NaCl per 1000 mg of ceftolozane, respectively). Comparison of the compositions are listed in Table 14. The amounts of the composition RT 63′, as measured by the HPLC method, are summarized in Table 15.

TABLE 14 Comparison of the CXA-201 Compositions Lot CXA-101 NaCl Tazobactam C1 10% High Na C2 20% Mid Na C3 20% Low Na C4 20% Mid Arginate C5 20% Low Arginate

TABLE 15 RT 63′ Peak Area at t = 3 months, 25° C./60% RH storage 1^(st) data 2nd data 3rd data collection collection collection Sample Summary RT Area % RT Area % RT Area % C1 High salt + 63.90 0.03 63.30 0.08 62.49 0.14 Tazo Na C2 Mid salt + 63.78 0.06 63.12 0.12 62.45 0.28 Tazo Na C3 Low salt + 63.75 0.12 63.11 0.14 62.46 0.29 Tazo Na C4 Mid salt + 63.76 0.10 63.16 0.13 62.44 0.28 Tazo Arg C5 Low salt + 63.72 0.08 63.14 0.16 62.46 0.33 Tazo Arg

Conclusion:

At the three month time point, the reduced salt formulations were observed to be not as stable as the full salt formulation; and trends indicate that reduction in salt causes at least 1.5-fold greater composition at RT=63 minutes, as measured by HPLC. The compositions comprising 480 mg NaCl per 1000 mg of ceftolozane had the least amount of the composition RT 63′ after 3 months at 25° C. The amount of the composition RT 63′ in the compositions comprising 125 mg NaCl per 1000 mg of ceftolozane was 1.5-fold or greater than the amount of the composition 63′ in the compositions comprising 480 mg NaCl per 1000 mg of ceftolozane. The amount of the composition RT 63′ in the compositions comprising 62.5 mg NaCl per 1000 mg of ceftolozane was 2-fold or greater than the amount of the composition RT 63′ in the compositions comprising 480 mg NaCl per 1000 mg of ceftolozane. Thus, the CXA-201 compositions comprising high amounts of sodium chloride (e.g., 125-1000 mg sodium chloride per 1000 mg of ceftolozane) were more chemically stable than the compositions comprising low amounts of sodium chloride (e.g., less than 125 mg sodium chloride per 1000 mg of ceftolozane).

B. Improvement in the Purity of Ceftolozane in CXA-201 Pharmaceutical Compositions with Varying Amounts of Sodium Chloride

A stability study was carried out at 30° C. and 60° C. analyzed by HPLC. The sodium chloride content in the CXA-201 compositions is described in Table 16. The HPLC data at 60° C. are summarized in Tables 17-20. The data are also plotted in FIGS. 8-11 to show the trends of the purity, and the amounts of the composition peak 1, the composition with a RRT of 0.43 and the composition peak 3, and the composition peak 7 in the CXA-201 compositions with respect to NaCl.

TABLE 16 The Sodium Chloride Content in the CXA-201 Compositions Samples NaCl content B1 481.0 mg sodium chloride per 1000 mg of ceftolozane B2 125.0 mg sodium chloride per 1000 mg of ceftolozane B3  75.0 mg sodium chloride per 1000 mg of ceftolozane B4  50.0 mg sodium chloride per 1000 mg of ceftolozane

TABLE 17 The Purity of Ceftolozane in CXA-201 Compositions with Varying Amounts of Sodium Chloride Day B1 B2 B3 B4 t0 0 98.1 97.8 97.8 97.7 1 day/60° C. 1 97.2 96.3 96.2 96.0 1 day/30° C. 1 98.2 97.7 97.6 97.6 3 days/60° C. 3 95.4 94.9 94.7 94.6 (Δ_(t0-t3)) (2.7) (2.9) (3.1) (3.1) 3 day/30° C. 3 98.0 97.5 97.4 97.3 7 days/60° C. 7 92.7 93.8 93.6 93.4 7 day/30° C. 7 97.8 97.2 97.0 96.9 Total Δ/60° C. 5.3 4.0 4.2 4.3 Total Δ/30° C. 0.3 0.6 0.8 0.8

TABLE 18 The HPLC Peak Area of Composition Peak 1 in CXA-201 Compositions with Varying Amounts of Sodium Chloride Day B1 B2 B3 B4 t0 0 0.47 0.38 0.38 0.41 1 day/60° C. 1 1 1.08 1.09 1.14 1 day/30° C. 1 0.48 0.44 0.45 0.49 3 days/60° C. 3 1.85 1.64 1.66 1.71 3 day/30° C. 3 0.53 0.53 0.56 0.61 7 days/60° C. 7 3.3 2.28 2.25 2.29 7 day/30° C. 7 0.62 0.67 0.71 0.77 INCREASE %/60° C. 2.83 1.9 1.87 1.88 INCREASE %/30° C. 0.15 0.29 0.33 0.36

TABLE 19 The Total HPLC Peak Area of the Composition with a RRT of 0.43 and Composition Peak 3 in CXA-201 Compositions with Varying Amounts of Sodium Chloride Day B1 B2 B3 B4 t0 0 0.15 0.12 0.12 0.12 1 day/60° C. 1 0.36 0.35 0.31 0.32 1 day/30° C. 1 0.13 0.12 0.13 0.12 3 days/60° C. 3 0.92 0.67 0.65 0.62 3 days/30° C. 3 0.17 0.16 0.17 0.16 7 days/60° C. 7 1.29 0.78 0.75 0.71 7 days/30° C. 7 0.19 0.19 0.20 0.20 INCREASE %/60° C. 1.14 0.66 0.63 0.59 INCREASE %/30° C. 0.04 0.07 0.08 0.08

TABLE 20 The HPLC Peak Area of Composition Peak 7 in CXA-201 Compositions with Varying Amounts of Sodium Chloride Day B1 B2 B3 B4 t0 0 0.69 1.01 1.01 1.01 1 day/60° C. 1 0.73 1.12 1.15 1.18 1 day/30° C. 1 0.68 1.00 0.99 0.95 3 days/60° C. 3 0.8 1.24 1.27 1.27 3 days/30° C. 3 0.68 1.00 1.01 1.03 7 days/60° C. 7 0.94 1.32 1.35 1.4 7 days/30° C. 7 0.68 1.02 1.05 1.06 INCREASE %/60° C. 0.25 0.31 0.34 0.39 INCREASE %/30° C. NC 0.01 0.04 0.05

Conclusion:

The stability data shows that high sodium chloride content enhances stability of CXA-201 compositions.

Similarly to CXA-101 compositions, CXA-201 compositions comprising high amounts of sodium chloride (e.g., 125-1000 mg sodium chloride per 1000 mg of ceftolozane) were found to be more chemically stable than CXA-201 compositions comprising low amounts of sodium chloride (e.g., less than 125 mg sodium chloride per 1000 mg of ceftolozane). Table 17 shows that, by day 3 of heating at 60° C., sample B1 containing the highest salt concentration is most stable, i.e., has the lowest Δ_(t0-t3) of all samples. By day 3, the sample with the lowest salt concentration, B4, has the highest Δ_(t0-t3) indicating the most degradation. Overall, B4 has degraded 15% more than B1.

Example 9 Manufacturing Process of a CXA-201 Composition (Comprising Tazobactam and Ceftolozane) by Blending A. Sterile Dry Blending of Bulk Lyophilized Ceftolozane and Bulk Lyophilized Tazobactam

A low energy drum blender that agitates the material by tumbling and also moving the bed up and down is used. A representative process of blending is described as follows, also shown in FIG. 1. The blender was charged with 23.4 kg of CXA-101 bulk product, and 5.4 kg of tazobactam bulk product. Both the CXA-101 and tazobactam were individually lyophilized beforehand. The material was blended for 180 minutes. In-process tests of content assay for both CXA-101 and tazobactam were performed to assess the homogeneity using the samples of blend materials taken from three places. The relative standard deviation (RSD) for each of CXA-101 and tazobactam content assay was no greater than 2% and the RSD for the ratio of CXA-101/tazobactam was no greater than 2% (See Table 21).

TABLE 21 In-Process Testing of Blending Samples of a CXA-201 Composition at Three Places Acceptance Limits Results (expected 60 120 180 Test value) Sampling minute minute minute Content: 30.4%-37.2% 1 34.24 34.07 34.42 Ceftolozane¹ 2 34.62 34.21 34.66 3 34.71 34.60 34.85 Mean ³ 34.52 34.30 34.64 RSD % 0.72 0.80 0.63 Content: 15.2%-18.6% 1 17.96 18.20 17.12 Tazobactam² 2 16.90 18.26 16.51 3 17.27 16.93 17.02 Mean ³ 17.38 17.80 16.89 RSD % 3.10 4.22 1.96 Ratio of Content 2.00⁴ 1 1.91 1.87 2.01 (w/w) 2 2.05 1.87 2.10 ceftolozane/ 3 2.01 2.04 2.05 tazobactam Mean ³ 1.99 1.93 2.05 RSD % 3.69 5.12 2.2 RSD = relative standard deviation ¹Theoretical value: 33.96% Acceptance limits are 90%-110% of the theoretical value. ²Theoretical value: 16.99% Acceptance limits are 90%-110% of the theoretical value. ³ Three samples are taken at each time point at three places to measure the percentage by weight of ceftolozane and tazobactam. The “Mean” is the average of the percentages or the weight ratios of Ceftolozane/tazobactam. ⁴Acceptance limits were established based on batch history. B. Packaging into Sterbags®

The blended powder is then discharged into Sterbags®.

C. Finished CXA-201 Drug Product

A fill and finish process is utilized for the final drug product, which is a pharmaceutical composition comprising CXA-101 and tazobactam at a ratio of 1000 mg/500 mg. Glass containers are washed with WFI and depyrogenated in a Class 100 depyrogenation tunnel at a temperature of 320° C. Pre-washed and pre-siliconized stoppers are autoclaved for 40 minutes at 121° C. The bulk drug product is packaged in a Sterbag® system comprised of three bags. The outer bag is cleaned with disinfectant in a Class 10,000 clean room. The bag system is placed in a pass-through UV box where it is subjected to UV radiation (>20 μW/cm²) for 20 minutes to sterilize the surface of the outer bag. The outer bag is removed and left in the UV box. The middle bag is placed in a Class A laminar airflow (LAF) hood. The sterile middle bag is removed under LAF. The sterile, bottle-shaped inner bag is then placed in a sterile stainless steel carrier and attached to the filling machine.

Sterile bulk CXA-101/tazobactam drug product is filled under a nitrogen blanket into 30-mL, Type I clear glass containers. The sterile drug product is gravity-fed into the filling machine under LAF. container fill weights are periodically checked throughout the filling operation to ensure proper operation of the filling line. Filling and stoppering operations are performed under Class 100 LAF conditions. Capping and container washing are done in the Class 10,000 clean room.

Example 10 Assessment of Blend Combination Drug Product A. Preparation of the Blend Combination Drug Product (CXA-201 Composition)

The blend drug product was prepared, as described above in Example 9, on lab scale using a small blender. The components of the blend composition are shown in Table 22.

TABLE 22 Components of the Blend Composition Quantity as active Component Composition components CXA-101 for Ceftolozane 10.8 g Injection Bulk L-Arginine 6.7 g (25 g) CXA-201 Comp. Citric acid 233 mg Sodium chloride 5.2 g Tazobactam 5.4 g sodium sterile (as Tazo free Bulk (6 g) acid)

B. Stressed Stability Test

Stability studies of this CXA-201 composition prepared by the blending process were carried out at 25° C. and 40° C. The composition was analyzed using HPLC method described in Example 1. The following Tables 23 and 24 are summaries of the HPLC measurements at time zero, after one month (T1), and after three months (T2).

TABLE 23 Stability Data of Blend CXA-201 Composition at 25° C./RH = 60% Test items Specifications T0 T1 25° C. T2 25° C. Related Substances Peak1 ≦1.50%   0.61%   0.93%   1.08% Peak2 ≦0.40% <0.03% <0.03% <0.03% Peak3 ≦0.30% <0.03% <0.03% <0.03% Peak4 ≦0.80%   0.03%   0.03%   0.04% Peak5 ≦1.00%   0.09%   0.12%   0.13% Peak6 ≦0.15% <0.03% <0.03% <0.03% Peak7 ≦2.00%   1.28%   1.34%   1.35% Peak8 ≦0.15% <0.03% <0.03% <0.03% Peak9 ≦0.60%   0.03% <0.03%   0.03% Peak10, 11 ≦0.30% <0.03%   0.04%   0.05% Sing. Unk. ≦0.15%   0.13%   0.13%   0.14% Total ≦5.00%   2.49%   3.03%   3.28% Assay CXA-101 Teor. % = 32.6%   32.5% n.a. n.a. Assay Tazobactam Teor. % = 17.4%    .2% n.a. n.a. Tazobactam Related  ≦4.0%   0.07%   0.12%   0.14% Compound A   K.F.  ≦4.0%    2.6% n.a. n.a. pH 5.0-7.0 6.0 5.6 5.1

TABLE 24 Stability Data of Blend CXA-201 Composition at 40° C./RH = 75% Test items Specifications T0 T1 40° C. T2 40° C. Related Substances Peak1 ≦1.50%   0.61%   1.66%   2.28% Peak2 ≦0.40% <0.03% <0.03% <0.03% Peak3 ≦0.30% <0.03% <0.03%   0.04% Peak4 ≦0.80%   0.03%   0.04%   0.05% Peak5 ≦1.00%   0.09%   0.13%   0.14% Peak6 ≦0.15% <0.03% <0.03% <0.03% Peak7 ≦2.00%   1.28%   1.41%   1.46% Peak8 ≦0.15% <0.03% <0.03% <0.03% Peak9 ≦0.60%   0.03% <0.03%   0.03% Peak10, 11 ≦0.30% <0.03%   0.08%   0.09% Sing. Unk. ≦0.15%   0.13%   0.14%   0.13% Total ≦5.00%   2.49%   4.21%   5.27% Assay CXA-101 Teor. % = 32.6%   32.5% n.a. n.a. Assay Tazobactam Teor. % = 17.4%   18.2% n.a. n.a Tazobactam Related  ≦4.0%   0.07%   0.35%   0.54% Compound A   K.F.  ≦4.0%    2.6% n.a. n.a. pH 5.0-7.0 6.0 5.0 4.4

C. Conclusion

The data at both 25° C. and at 40° C. have shown that the blending process completely inhibits formation of the compound RRT=1.22.

Example 11 Alkalizing Agent Selection

Compositions for intravenous administration should be formulated to resemble the and pH of human blood to reduce vascular complications. The recommended pH is between 5 and 9 (ideal pH is as close to 7.4 as possible). Departing from this recommended pH ranges of an intravenously administered composition can result in the development of complications such as phlebitis, or inflammation of the veins. Marc Stranz, A Review of pH and Osmolarity, 6 Int'l J. of Pharm. Compounding 216, 218 (May/June 2002). Unfortunately, few drug infusions are stable at a suitable pH for intravenous administration. Depending on the molecular structure, a drug is most stable or has the best solubility at a particular pH range (e.g., pH<6) and divergence from this pH range may lead to increased drug decomposition. It is thus challenging to find a balance between the safe limits of pH and optimum drug stability in compositions for intravenous administration. Marc Stranz, The Implications of Osmolality, Osmolarity and pH in Infusion Therapy, INS Annual Conference (May 2005).

A formulation close to physiologic pH was targeted. This necessitates an alkalizing agent due to intrinsic pH 1.92 of ceftolozane in solution (2%). The initial study of alkalizing agents included sodium hydroxide, L-arginine, tris, sodium bicarbonate, meglumine, diethanolamine, and triethanolamine. Samples containing 100 mg ceftolozane sulfate, 22.9 mg sodium chloride, 200 mg maltose, and 2 mg citric acid anhydrous were prepared and adjusted to ˜pH 4. The samples were lyophilized and powders stored at 70° C. for 3 days, 60° C. for 3, 6 and 9 days and at 40° C. for one month. The stored samples were then analyzed for ceftolozane content. Results are reported below in Table 25:

TABLE 25 Effect of Alkalizing Agent on Ceftolozane Recovery Storage Sodium hydroxide L-arginine Tris Sodium bicarbonate Meglumine Diethanolamine Triethanolamine 70° C., 3 d 93.3 93.0 83.1 93.8 71.2 52.7 28.0 60° C., 3 d 97.0 96.3 93.5 93.9 94.4 91.6 67.2 60° C., 6 d 95.7 95.5 89.8 96.0 89.8 83.6 59.0 60° C., 9 d 93.9 93.1 87.5 93.8 88.7 82.0 75.9 40° C./75% RH, 1 mo 97.3 97.0 95.1 97.6 97.6 94.4 94.4

Ceftolozane recovery was consistently above 90% in the presence of sodium hydroxide, L-arginine, or sodium bicarbonate. Although sodium hydroxide performed well, as a strong base, it could promote base hydrolysis of the active more readily during scale up and be more difficult to dry during lyophilization than other alkalizing agents. Accordingly sodium hydroxide was not considered for further formulation development. L-arginine was thus chosen as the alkalizing agent for the formulation.

To ensure suitability of L-arginine as an alkalizing agent, a study was conducted to compare L-arginine against sodium bicarbonate. In this study, solutions were prepared to contain ceftolozane in the presence of sodium chloride and citric acid adjusted to approximately pH 6 with either L-arginine or sodium bicarbonate. The solutions were then lyophilized and samples distributed for accelerated and regular storage. A summary of the total additional compounds and pH for the various conditions after 1 month is presented in Table 26.

TABLE 26 Effect of L-Arginine and Sodium Bicarbonate on Ceftolozane Related Substances during Storage, pH 6 Bulk solution 632 mg L-arginine 288 mg sodium bicarbonate composition per 485 mg sodium chloride 481 mg sodium chloride 1000 mg 21 mg citric acid 21 mg citric acid ceftolozane Total Total free base Related Sample Related Sample Storage condition Substances pH Substances pH Initial 1.42% 5.8 2.12% 5.8 5° C., 1 month 1.38% 5.8 2.66% 5.6 25° C., 1 month 1.74% 5.5 4.99% 4.8 40° C., 1 month 2.32% 5.0 5.93% 4.5

As seen in the table the bicarbonate-adjusted sample showed a larger increase in related substances and a less stable pH profile. Accordingly, it was decided to maintain L-arginine as the alkalizing agent in the formulation.

Example 12 Components of a CXA-201 Composition

An example of a batch formulae for ceftolozane composition (compounding of ceftolozane substance with excipients such as citric acid, sodium chloride, and L-arginine followed by sterile lyophilization) is found below in Table 27.

TABLE 27 Batch Formula for Ceftolozane composition Target Composition Amount per Batch (kg) Component mg/g 1 2 Ceftolozane Sulfate^(i)) 172.1 114.0 202.6 Citric Acid, Anhydrous, USP 3.2 2.1 3.7 Sodium Chloride, USP 73.1 48.3 86.0 L-Arginine, USP ~90 59.7 106.0 QS to achieve target pH^(ii)) Water for Injection, USP QS to 1000 QS QS Total Batch Size 660 1175 1) Ceftolozane sulfate is charged based on its measured potency to obtain 150 mg free base/g solution. 2) L-arginine is added as needed to obtain pH 6.5 ± 0.5 in the bulk solution; 90 mg per gram solution is considered a representative amount.

An example of a batch formula for the ceftolozane/tazobactam drug product is presented in Table 28 below.

TABLE 28 Batch Formula Ceftolozane/Tazobactam Drug Product Amount per Amount per Batch, Component container, mg kg Ceftolozane 2255 112.8 composition^(i)) Tazobactam^(ii)) 537 26.9 Nitrogen, NF^(iii)) — — Total 2792 139.7 Total Batch Size, kg 139.7 Total container Quantity 50,000 composition^(i))The target fill for ceftolozane is 1000 mg free base, added to the container as the composition. The amount 2255 mg is based on 100% theoretical potency of the composition. Actual weight will vary based on composition measured potency. ^(ii))The target fill for tazobactum is 500 mg free acid, added to the container as its sodium salt form. The amount 537 mg is based on 100% theoretical potency. ^(iii))Nitrogen is used as a processing aid to blanket containers after powder filling and prior to insertion of stopper.

The unit composition of a dosage for reconstitution is described in Table 29.

TABLE 29 Unit Compositions of Ceftolozane/Tazobactam for Injection, 1000 mg/500 mg Nominal Composition mg per Component Function container Ceftolozane Ceftolozane Active 1147 composition¹⁾ Sulfate Citric Acid, Chelating  21 Anhydrous Agent Sodium Stabilizing  487 Chloride Agent L-Arginine Alkalizing  600²⁾ Agent Q.S. for pH adjustment Tazobactam Sodium³⁾ Active  537 Nitrogen Processing Q.S. Aid^((a)) Total Weight 2792 ¹⁾Actual amount of ceftolozane composition will vary based on the measured potency. Ceftolozane sulfate, 1147 mg, corresponds to 1000 mg ceftolozane free base. ²⁾L-arginine is added as needed to achieve pH 6.5 ± 0.5; 600 mg per container is considered a representative total amount. ³⁾Actual weight of tazobactam sodium will vary based on the measured potency. Tazobactam sodium 537 mg, corresponds to 500 mg tazobactam free acid 4) Nitrogen blanket is applied after powders are dispensed to the container and prior to insertion of stopper.

Example 12a Development and Implementation of a System to Prevent Cross-Contamination in Accordance with FDA Guidance

A recently published (April 2013) Food and Drug Administration Guidance for Industry Non Penicillin Beta-Lactam Drugs: A CGMP Framework for Preventing Cross-Contamination provides direction on prevention of cross-contamination for facilities that manufacture non-penicillin beta-lactam drugs. Provided herein are steps for the development and implementation of a system to prevent cross-contamination due to the introduction of both sterile ceftolozane drug product intermediate and tazobactam sodium into a facility that is in conformance with FDA Guidance.

Segregation steps to conform with FDA Guidance can include, but are not limited to:

-   -   Relocation all other drug products to other sites     -   Separating the ceftolozane/tazobactam product filling line and         the veterinary cephapirin product filling line     -   Creating separate HVAC systems     -   Establishing separate warehouse areas     -   Formalizing separate material, waste and personnel flows     -   Constructing temporary facilities for gowning and entrance to         the line used for the ceftolozane/tazobactam drug product.     -   Constructing new walls, modifying and reinforcing existing walls     -   Equipping the existing emergency egress with alarms and gaskets         to completely separate both lines throughout all the floors of         the building     -   Creating the permanent separation of locker, rest and break         rooms for both lines of the facility:     -   Dedicated maintenance and operations personnel for each part of         the facility including different uniform colors for each part of         the facility     -   Dedicated equipment and tools for each part of the facility     -   An Emergency Recovery plan

Example 13 Physicochemical and Biological Properties Ceftolozane/Tazobactam for Injection, 1000 mg/500 mg

As a product intended for intravenous use, several properties are important for physiological compatibility. These include particulate matter, sterility, endotoxin limit, pH, and osmolality. Particulate matter and sterility are controlled at the point of manufacture. The drug product is processed aseptically throughout the entire manufacturing process, inclusive of ceftolozane, tazobactam sodium, and ceftolozane/tazobactam in-container drug product.

The ceftolozane/tazobactam drug product is controlled to approximately pH 6, to provide physiological comfort, while still assuring adequate stability for the drug substances. The ceftolozane drug product intermediate is controlled during compounding to pH 6.5±0.5 and is controlled at release to pH 5 to 7. The tazobactam sodium is controlled at release to pH 5 to 7.

Ceftolozane/tazobactam following reconstitution with normal saline and dilution for infusion also in normal saline (10 mg/mL ceftolozane; 5 mg/mL tazobactam) is slightly hypertonic, with osmolality approximately 500 mOsm/kg. However, slightly hypertonic intravenous infusion solutions are not uncommon as drug products are commonly prepared and diluted with already-isotonic solutions, such as normal saline. The generally accepted maximum upper limit for peripheral intravenous administration is approximately 900 mOsm/kg, though admixtures 600 to 900 mOsm/kg are typically administered through a central line. Therefore, to be within the limits of this range, the infusion product is less than 600 mOsm/kg.

Example 14 Determining Osmolality of CXA-201 Compositions

CXA-101 and Tazobactam Sodium samples (#1-#3) were reconstituted as follows:

-   Sample#1: Weighed 0.103 g of Tazobactam Sodium and 0.462 g of     CXA-101dissolved in 4 mL of WFI Water and 6 mL of USP Normal Saline. -   Sample#2: Weighed 0.103 g of Tazobactam Sodium and 0.462 g of     CXA-101dissolved in 4 mL of WFI Water added 10 mL of USP Normal     Saline. -   Sample#3: Weighed 0.103 g of Tazobactam Sodium dissolved in 1 mL of     WFI Water and 0.462 g of CXA-101dissolved in 1 mL of WFI Water then     mixed together added 10 mL of USP Normal Saline. -   Tazobactam Sodium (Potency: 97.5%) -   CXA-101 (Potency: 43.3%) -   WFI Water -   USP Normal Saline

The osmolality of CXA-101 and Tazobactam Sodium samples (#1-#3) was then determined using a freezing point depression Osmometer (available from Advanced Instruments, Inc.).

TABLE 30 Osmolality of Reconstituted Solutions CXA- Tazo- 101 bactam Osmol- Conc. Sodium atity Sample (mg/ Conc. (mOsm/ # mL) (mg/mL) WFI Water Saline kg) 1 20.0 10.0 40% (40 mL) 60% (60 mL) 589 2 14.3 7.1 29% (40 mL) 71% (100 mL) 512 3 16.7 8.3 17% (20 mL) 83% (100 mL) 604

A unit dosage form composition of Table 29 was reconstituted with 10 mL of Sterile WFI or USP Normal Saline then added into 100 mL 5% Dextrose Injection (D5W) or 0.9% Sodium Chloride (NS) bags and the osmolality of the resulting bag solution was determined as shown in table Table 30a below.

TABLE 30a Osmolarity of Ceftolozane Bag Solution (mOsm/kg) Time Point sWFI-D5W NS-D5W sWFI-NS NS-NS RT T0 446 470 449 478

In Table 30a, data for osmolality of the following product reconstitution scenarios was determined using the composition from Table 29

-   -   5% Dextrose Injection USP, 100 mL Bag (Baxter)     -   0.9% Sodium Chloride Injection USP, 100 mL Bag (Baxter)     -   sWFI—D5W: reconstituted with Sterile WFI then added into 5%         Dextrose Injection bag     -   NS—D5W: reconstituted with USP Normal Saline then added into 5%         Dextrose Injection bag     -   sWFI—NS: reconstituted with Sterile WFI then added into 0.9%         Sodium Chloride Injection Bag     -   NS—NS: reconstituted with USP Normal Saline then added into 0.9%         Sodium Chloride Injection bag

Example 15 Excipients in Ceftolozane Drug Product Intermediate

The excipients in exemplary ceftolozane compositionswere chosen to ensure stability and processability of the ceftolozane drug substance into the drug product. The specific excipients, their quantities and functions are provided in Table 31. All excipients are compendial and typical for sterile pharmaceutical dosage forms, requiring no additional treatment prior to use in the formulation. The excipients are used in levels within the range established in other FDA approved products as described in the Inactive Ingredients Database (IID).

TABLE 31 Excipients Used in Ceftolozane Composition Inactive Amount, Concentration Ingredients mg/ in Infusion Rationale Database Component Function container Solution, % for Inclusion (IID) Range Citric Chelating  21 0.02 Used to 0.0025 to 50% acid agent prevent discoloration and degradation Sodium Stabilizing 487 0.49 Used as a stabilizing 0.187 to 45% Chloride agent agent for ceftolozane sulfate L-arginine Alkalizing 600^((a)) 0.60 Used to adjust 0.29 to 88% agent Q.S. for pH ceftolozane solution pH adjustment ^((a))L-arginine is added as needed to achieve pH 6.5 ± 0.5; 600 mg per container is considered a representative total amount.

Example 16 Manufacturing Process of a CXA-201 Composition (Comprising Tazobactam and Ceftolozane) by Co-Filling

The ceftolozane/tazobactam finished drug product is a sterile powder fill of lyophilized active ingredients ceftolozane drug product intermediate (composition) and tazobactam sodium together into a sterile single container. The lyophilized form of the sterile tazobactam sodium contains no excipients. Ceftolozane sulfate drug substance is converted first into a sterile drug product intermediate, composition, by formulation with citric acid, sodium chloride and L-arginine, followed by lyophilization.

The full manufacturing process includes unit operations typical of an aseptic lyophilization process and aseptic powder filling process. The overall process can be outlined in two stages, as presented in the manufacturing flow chart of FIG. 12. The first stage is the manufacturing of the sterile ceftolozane composition. The second stage is the filling of the sterile drug powders into containers for the final drug product. The major process steps are:

Preparation of the sterile ceftolozane composition comprises

-   -   compounding the bulk solution for lyophilization;     -   sterile filtering the bulk solution;     -   lyophilizing the bulk solution into bulk powder;     -   grinding and sieving of the sterile bulk powder; and     -   aseptic packaging of the sterile bulk powder in Sterbags®.

Filling of the sterile bulk powders comprises

-   -   receipt of ceftolozane and tazobactam sterile powders at site;     -   aseptic filling both sterile powders into the container         sequentially;     -   blanketing the container with a nitrogen headspace;     -   stoppering and crimping the container; and     -   inspecting the container prior to secondary packaging. 

1-26. (canceled)
 27. A solid pharmaceutical composition comprising ceftolozane or a pharmaceutically acceptable salt thereof and tazobactam sodium, said composition comprising less than 0.1% by weight of a compound of formula (III)

when stored for 1 month at a temperature of 25° C. and at a relative humidity of 60%, wherein the ceftolozane or pharmaceutically acceptable salt thereof and tazobactam sodium provide ceftolozane active and tazobactam active in a ratio of 2:1 by weight.
 28. The composition of claim 27, wherein the pharmaceutically acceptable salt of ceftolozane is ceftolozane sulfate.
 29. The composition of claim 48, which is provided in a unit dosage form.
 30. The composition of claim 48, which contains less than 0.05% by weight of the compound of formula (III) when stored for 1 month at a temperature of 25° C. and at a relative humidity of 60%.
 31. The composition of claim 48, which contains less than 0.03% by weight of the compound of formula (III) when stored for 1 month at a temperature of 25° C. and at a relative humidity of 60%.
 32. The composition of claim 48, wherein the compound of formula (III) is not present when stored for 1 month at a temperature of 25° C. and at a relative humidity of 60%.
 33. The composition of claim 48, wherein the amount of ceftolozane active is 1,000 mg and the amount of tazobactam active is 500 mg.
 34. The composition of claim 48, wherein the amount of ceftolozane active is 2,000 mg and the amount of tazobactam active is 1,000 mg.
 35. The pharmaceutical composition of claim 48, comprising 1147 mg ceftolozane sulfate and 537 mg tazobactam sodium.
 36. An injectable solution comprising a pharmaceutical composition of claim 48 dissolved in a pharmaceutically acceptable vehicle.
 37. The injectable solution of claim 56, wherein the pharmaceutically acceptable vehicle is selected from water, 0.9% sodium chloride and 5% Dextrose.
 38. A pharmaceutical composition comprising ceftolozane or a pharmaceutically acceptable salt thereof and tazobactam sodium, said composition comprising less than 0.1% of a material having a mass spectra depicted in FIG. 14, wherein the ceftolozane or pharmaceutically acceptable salt thereof and tazobactam sodium provide ceftolozane active and tazobactam active in a ratio of 2:1 by weight.
 39. The composition of claim 58, wherein the pharmaceutically acceptable salt of ceftolozane is ceftolozane sulfate.
 40. The composition of claim 59, which is provided in a unit dosage form.
 41. The composition of claim 59, which contains less than 0.05% by weight of the material with the mass spectra depicted in FIG. 14 when stored for 1 month at a temperature of 25° C. and at a relative humidity of 60%.
 42. The composition of claim 59, which contains less than 0.03% by weight of the material with the mass spectra depicted in FIG. 14 when stored for 1 month at a temperature of 25° C. and at a relative humidity of 60%.
 43. The composition of claim 48, wherein the material with the mass spectra depicted in FIG. 14 is not present when stored for 1 month at a temperature of 25° C. and at a relative humidity of 60%.
 44. The composition of claim 59, wherein the amount of ceftolozane active is 1,000 mg and the amount of tazobactam active is 500 mg.
 45. The composition of claim 59, wherein the amount of ceftolozane active is 2,000 mg and the amount of tazobactam active is 1,000 mg.
 46. The pharmaceutical composition of claim 59, comprising 1147 mg ceftolozane sulfate and 537 mg tazobactam sodium.
 47. An injectable solution comprising a pharmaceutical composition of claim 59 dissolved in a pharmaceutically acceptable vehicle.
 48. The injectable solution of claim 67, wherein the pharmaceutically acceptable vehicle is selected from water, 0.9% sodium chloride and 5% Dextrose. 